Broadly neutralizing antibodies directed against the rabies virus glycoprotein and uses thereof

ABSTRACT

The invention relates to antibodies, and antigen binding fragments thereof, that potently neutralize infection of both RABV and non-RABV lyssaviruses. The invention also relates to antigenic sites to which the antibodies and antigen binding fragments bind, as well as to nucleic acids that encode and immortalized B cells and cultured plasma cells that produce such antibodies and antibody fragments. In addition, the invention relates to the use of the antibodies and antibody fragments of the invention in screening methods as well as in the diagnosis, prophylaxis and treatment of RABV infection and infection with non-RABV lyssaviruses.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 470082_404 D1_SEQUENCE_LISTING.txt. The textfile is 70.5 KB, was created on May 22, 2020, and is being submittedelectronically via EFS-Web.

The present invention relates to antibodies, and antigen bindingfragments thereof, that potently neutralize infection of both rabiesvirus (RABV) and non-RABV lyssaviruses. The invention also relates toantigenic sites to which the antibodies and antigen binding fragmentsbind, as well as to nucleic acids that encode and immortalized B cellsand cultured plasma cells that produce such antibodies and antibodyfragments. In addition, the invention relates to the use of theantibodies and antibody fragments of the invention in screening methodsas well as in the diagnosis, prophylaxis and treatment of RABV infectionand infection with non-RABV lyssaviruses.

Rabies is a viral infection, that causes acute inflammation of thebrain. Rabies is distributed nearly worldwide and affects principallywild and domestic animals, but also involves humans, resulting in adevastating disease, which is nearly 100% invariably fatal inindividuals who do not receive post-exposure prophylaxis (PEP). Earlysymptoms of rabies can include fever and tingling at the site ofexposure. These symptoms are followed by one or more of the followingsymptoms: violent movements, uncontrolled excitement, fear of water, aninability to move parts of the body, confusion, and loss ofconsciousness. After symptoms appear, rabies almost always results indeath. The time period between contracting the disease and the start ofsymptoms is usually one to three months. However, this time period canvary from less than one week to more than one year. The time perioddepends on the distance the virus must travel to reach the centralnervous system.

Rabies is caused by a number of lyssaviruses including rabies virus andother lyssaviruses, for example European bat lyssavirus.

Lyssaviruses have helical symmetry, with a length of about 180 nm and across-section of about 75 nm. These viruses are enveloped and have asingle-stranded RNA genome with negative sense. The genetic informationis packed as a ribonucleoprotein complex in which RNA is tightly boundby the viral nucleoprotein. The RNA genome of the virus encodes fivegenes whose order is highly conserved: nucleoprotein (N), phosphoprotein(P), matrix protein (M), glycoprotein (G), and the viral RNA polymerase(L).

The Lyssavirus genus is subdivided into three phylogroups. Phylogroup Iincludes the species Rabies virus (RABV), European bat lyssavirus type 1(EBLV-1) and type 2 (EBLV-2), Duvenhage virus (DUVV), Australian batlyssavirus (ABLV), Aravan virus (ARAV), Khujand virus (KHUV), Bokelohbat lyssavirus (BBLV) and Irkut virus (IRKV). Phylogroup II includesLagos bat virus (LBV), Mokola virus (MOKV), and Shimoni bat virus (SHIVor SHIBV). The remaining viruses, West Caucasian bat virus (WCBV) andIkoma lyssavirus (IKOV) cannot be included in either of thesephylogroups but are related phylogenetically to each other, however,their genetic distances are greater than the distances betweenphylogroups I and II (Bourhy, H., et al. Journal of ClinicalMicrobiology 30, 2419-2426, 1992; Bourhy, H., et al. Virology 194,70-81, 1993; Amengual, B., et al. J Gen. Virol 78, 2319-2328, 1997;Hooper, P. T. et al. Bulletin de l'Institut Pasteur 95, 209-218, 1997;Kuzmin, I. V. et al. Virus Res 149, 197-210, 2010; Badrane, H., et al. JVirol 75, 3268-3276, 2001; Marston, D. A. et al. Emerg Infect Dis 18,664-667, 2012). Importantly, all genotypes of these lyssaviruses havecaused human and/or animal deaths in nature (Badrane, H., et al. J Virol75, 3268-3276, 2001).

Rabies virus (RABV) was the first of the fourteen lyssavirus genotypesto be identified. The rabies virus is a large bullet-shaped, enveloped,single stranded RNA virus classified and the genome of rabies viruscodes for five viral proteins: RNA-dependent RNA polymerase (L); anucleoprotein (N); a phosphorylated protein (P); a matrix protein (M)located on the inner side of the viral envelope; and an external surfaceglycoprotein (G). The G protein (62-67 kDa) is a type-I glycoproteincomposed of 505 amino acids that has two to four potentialN-glycosylation sites. The G protein covers the outer surface of thevirion envelope and is the only target antigen, which is able to inducevirus-neutralizing antibodies.

Rabies is widespread across the globe and approximately 10 millionpeople a year are treated after exposure to rabies, usually following abite from infected animals (dogs, bats, foxes, cats, monkeys raccoons,skunks, cattle, wolves, coyotes and others domestic and wild animals).Some 40,000 to 70,000 people are estimated to die of the disease eachyear, mainly in Africa, China and India, and 50% cases of rabiesworldwide occur in children. These data highlight the significant unmetmedical need for a safe, effective and affordable rabies treatment.

Rabies prevention is achieved either by pre- or post-exposurevaccination, mostly using modern, tissue culture-based vaccines.Immunizing before exposure (Pre-exposure prophylaxis (PrEP)) isrecommended for those who are at high risk and is achieved byadministration of a rabies vaccine (active immunization). The high-riskgroup includes people who work with bats or who spend prolonged periodsin areas of the world where rabies is common. Furthermore, theanti-rabies vaccine is recommended for people travelling to countries inAfrica and Asia, where rabies is endemic.

Currently available rabies vaccines include the most widely used buthighly risk-prone nerve tissue vaccines, or the safer but more costlycell culture and embryonated egg vaccines (CCEEVs). In Germany e.g. onlytwo anti-rabies vaccines are on the market, Rabipur® and“Tollwut-Impfstoff (human diploid cell [HDC]) inaktiviert”. Thesevaccines contain inactivated rabies virus. Both vaccines are recommendedfor pre- and postexposure use.

After exposure to the virus, a post-exposure prophylaxis (PEP) with therabies vaccine and a rabies immunoglobulin (RIG) are the standardtreatment preventing the disease, if the person receives the treatmentas early as possible after infection, i.e. during the first days afterthe infection. If left untreated until the start of the symptoms, rabiesis nearly 100% fatal. Thus, currently, there is no treatment for rabies.

The currently used “treatment” when someone is assumed to be infected bythe virus is post-exposure prophylaxis (PEP), which combines rabiesimmunoglobulin (RIG), in particular human or equine rabiesimmunoglobulins (HRIG and ERIG, respectively), with a rabies vaccine. Inparticular, patients receive one dose of RIG (passive immunization) andseveral doses of rabies vaccine (active immunization) according to theinformation of the rabies vaccine manufacturer. In a widely usedstandard therapy, for example, five doses of the vaccine areadministered over a twenty-eight day period, i.e. the first dose ofrabies vaccine is given as soon as possible after exposure, preferablyday 0, with additional doses on days 3, 7, 14, and 28 after the first(cf.rki.de/DE/Content/Infekt/EpidBull/Merkblaetter/Ratgeber_Tollwut.html,retrieved at Nov. 12, 2014). In contrast, rabies immunoglobulin (RIG)for passive immunization is administered only once, preferably at, or assoon as possible after, the initiation of post-exposure vaccination. Thedose of human rabies immunoglobulin (HRIG) proposed by the WHO is 20IU/kg body weight; for equine immunoglobulin (ERIG) and F(ab′)2 productsit is 40 IU/kg body weight (cf.who.int/rabies/human/WHO_strategy_prepost_exposure/en/indexi.html#,retrieved at Nov. 12, 2014). In particular, higher doses can reducevaccine efficacy. All of the rabies immunoglobulin, or as much asanatomically possible to avoid possible compartment syndrome, should beadministered into or around the wound site or sites. The remainingimmunoglobulin, if any, should be injected intramuscularly at a sitedistant from the site of vaccine administration. Rabies immunoglobulinmay be diluted to a volume sufficient for all wounds to be effectivelyand safely infiltrated (cf.who.int/rabies/human/WHO_strategy_prepost_exposure/en/indexi.html#,retrieved at Nov. 12, 2014). This is usually successful if administeredup to 24-48 hours following exposure. The HRIG is widely used,especially in developed countries, and is considered safer than ERIG.The high cost of HRIG and its limited availability prohibit its wide usein developing countries. Moreover, the vaccine and HRIG or ERIG do noteffectively protect against infection with different lyssavirus species(protection is inversely related to the genetic distance with thevaccine strain). Thus, the need to replace HRIG with at least an equallypotent and a safer rabies antibody-based product is considered to beimportant to improve the access to rabies biologicals, in particular indeveloping countries.

To this end, mouse monoclonal antibodies as well as human monoclonalantibodies have been developed in the last decade with two products inadvanced clinical trials. Namely, CL184 (produced by Crucell), which isa cocktail of two human antibodies called CR57 and CR4098, was developedto replace HRIGs in clinical trials up to phase III (Bakker, A. B. H. etal., J Virol 79, 9062-9068, 2005; Goudsmit J, Marissen W E, Weldon W C,Niezgoda M, Hanlon C A, Rice A B, Kruif J, Dietzschold B, Bakker A B,Rupprecht C E (2006) Comparison of an anti-rabies human monoclonalantibody combination with human polyclonal anti-rabies immune globulin.J Infect Dis 193: 796-801). However, recently the trial was stoppedbecause of the lack of neutralizing activity of the cocktail, or one ofthe two antibodies of the cocktail, against some circulating RABVisolates. Another human monoclonal antibody, which is presently testedin clinical phase III in India is RAB1, which is produced by MassBiologics and Serum Institute of India and which is based on a singlemonoclonal antibody (Sloan S E, Hanlon C, Weldon W, Niezgoda M, BlantonJ, Self J, Rowley K J, Mandell R B, Babcock G J, Thomas W D, Jr, et al(2007) Identification and characterization of a human monoclonalantibody that potently neutralizes a broad panel of rabies virusisolates. Vaccine 25: 2800-2810; Nagarajan T, Marissen W E, Rupprecht CE (2014) Monoclonal antibodies for the prevention of rabies: theory andclinical practice. Antibody Technology Journal 4: 1-12). However, RABVisolates that are not neutralized by each of these monoclonal antibodies(CR57, CR4098 and RAB1) have been identified (Kuzimina N A, Kuzmin I V,Ellison J A, Rupprecht C E (2013) Conservation of binding epitopes formonoclonal antibodies on the rabies virus glycoprotein. Journal ofAntiviral and Antiretrovirals 5: 37-43, Marissen W E, Kramer R A, RiceA, Weldon W C, Niezgoda M, Faber M, Slootstra J W, Meloen R H,Clijsters-van der Horst M, Visser T J, et al (2005) Novel rabiesvirus-neutralizing epitope recognized by human monoclonal antibody: finemapping and escape mutant analysis. J Virol 79: 4672-4678). In the caseof the RAM antibody, two out of 25 isolates tested were not neutralizedand three were poorly neutralized (Sloan S E, Hanlon C, Weldon W,Niezgoda M, Blanton J, Self J, Rowley K J, Mandell R B, Babcock G J,Thomas W D, Jr, et al (2007) Identification and characterization of ahuman monoclonal antibody that potently neutralizes a broad panel ofrabies virus isolates. Vaccine 25: 2800-2810). In this case the risk ofPEP failure is, at least in principle, higher than in the case of theCR57 and CR4098 antibody cocktail. These studies indicate that CR4098and RAM have a limited breadth of reactivity towards non-RABV isolatesand that a significant fraction of the RABV isolates tested are not oronly poorly neutralized by these antigenic site III antibodies. Indeed,for the lack of large RABV coverage the development of CL184 wasrecently halted, while RAM in still under development in a Phase 2/3 inIndia.

Accordingly, there is still a need to replace HRIG with an at leastequally potent and safer antibody-based product. Moreover, there is aneed for a product capable of preventing as well as treating orattenuating infection with different lyssaviruses with high potency andefficacy, i.e. a product which is not limited to neutralize RABV only,in particular since in some countries, e.g. in Europe and Australia,rabies is mainly transmitted by bats. Further, it is important to haveantibodies that target different epitopes and different antigenic siteson the various strains in order to avoid appearance of resistant virusstrains and to prevent the escape of resistant variants of the virus.

Moreover, since there is currently no treatment for rabies, there is aneed of a product which is effective in treating or attenuatinginfection, even if the exposure to the virus was more than 24 to 48hours before the first treatment with the product. The development ofsuch a treatment would be of benefit in particular for at least twoclasses of patients: those with known exposure to RABV but who havefailed to receive prompt post-exposure prophylaxis due to circumstancesand who are at increased risk of developing RABV infection, and thosewho did not recognize contact with the virus and present signs (ofdifferent severity) of the disease (e.g. individuals infected byunnoticed contacts with infected bats; RABV of bat origin where dograbies is controlled has become the leading cause of human rabies). Thedevelopment of a product of potent and broadly neutralizing antibodiesmay thus help to expand the post-exposure treatment window for humanRABV infection, that is currently limited to the first days afterinfection. In these individuals the RABV might has already reached theCNS tissues and early or late signs of the disease might have alsoappeared. These patients could benefit from a treatment with highlypotent neutralizing antibodies that can leak across the blood brainbarrier (or administered directly in the CSN) delivering a sufficientamount of antibodies capable of effectively neutralizing the virusreplication in the CNS tissue.

In view of the above, it is the object of the present invention toprovide an antibody-based product, which is at least equally potent, butsafer and more cost-effective compared to HRIG. Moreover, it is theobject of the present invention to provide a product which is capable ofpreventing as well as treating or attenuating infection with differentlyssaviruses with high potency and efficacy, i.e. a product which is notlimited to neutralize RABV only. Furthermore, it is the object of thepresent invention to provide a product which is effective in treating orattenuating infection, even if the exposure to the virus was more than24 to 48 hours before the first treatment with the product. In summary,it is the object of the present invention to provide improvedantibodies, or antigen binding fragments thereof, as well as relatednucleic acid molecules, vectors and cells and pharmaceuticalcompositions, which overcome the above discussed disadvantages of theprior art by a cost-effective and straight-forward approach.

The object underlying the present invention is solved by the claimedsubject matter.

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isnot intended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the term “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step but not the exclusion of any othernon-stated member, integer or step. The term “consist of” is aparticular embodiment of the term “comprise”, wherein any othernon-stated member, integer or step is excluded. In the context of thepresent invention, the term “comprise” encompasses the term “consistof”. The term “comprising” thus encompasses “including” as well as“consisting” e.g., a composition “comprising” X may consist exclusivelyof X or may include something additional e.g., X+Y.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means x±10%.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the human or animal body or of one of its parts thatimpairs normal functioning, is typically manifested by distinguishingsigns and symptoms, and causes the human or animal to have a reducedduration or quality of life.

As used herein, reference to “treatment” of a subject or patient isintended to include prevention, prophylaxis, attenuation, ameliorationand therapy. The terms “subject” or “patient” are used interchangeablyherein to mean all mammals including humans. Examples of subjectsinclude humans, cows, dogs, cats, horses, goats, sheep, pigs, andrabbits. In one embodiment, the patient is a human.

As used herein, the terms “antigen binding fragment,” “fragment,” and“antibody fragment” are used interchangeably to refer to any fragment ofan antibody of the invention that retains the antigen-binding activityof the antibody. Examples of antibody fragments include, but are notlimited to, a single chain antibody, Fab, Fab′, F(ab′)₂, Fv or scFv.Further, the term “antibody” as used herein includes both antibodies andantigen binding fragments thereof.

As used herein, a “neutralizing antibody” is one that can neutralize,i.e., prevent, inhibit, reduce, impede or interfere with, the ability ofa pathogen to initiate and/or perpetuate an infection in a host. Theterms “neutralizing antibody” and “an antibody that neutralizes” or“antibodies that neutralize” are used interchangeably herein. Theseantibodies can be used alone, or in combination, as prophylactic ortherapeutic agents upon appropriate formulation, in association withactive vaccination, as a diagnostic tool, or as a production tool asdescribed herein.

Doses are often expressed in relation to the bodyweight. Thus, a dosewhich is expressed as [g, mg, or other unit]/kg (or g, mg etc.) usuallyrefers to [g, mg, or other unit] “per kg (or g, mg etc.) bodyweight”,even if the term “bodyweight” is not explicitly mentioned.

The term “specifically binding” and similar reference does not encompassnon-specific sticking.

The term “vaccine” as used herein is typically understood to be aprophylactic or therapeutic material providing at least one antigen,preferably an immunogen. The antigen or immunogen may be derived fromany material that is suitable for vaccination. For example, the antigenor immunogen may be derived from a pathogen, such as from bacteria orvirus particles etc., or from a tumor or cancerous tissue. The antigenor immunogen stimulates the body's adaptive immune system to provide anadaptive immune response. In particular, an “antigen” or an “immunogen”refers typically to a substance which may be recognized by the immunesystem, preferably by the adaptive immune system, and which is capableof triggering an antigen-specific immune response, e.g. by formation ofantibodies and/or antigen-specific T cells as part of an adaptive immuneresponse. Typically, an antigen may be or may comprise a peptide orprotein which may be presented by the MHC to T-cells.

As used herein, “sequence variant” refers to any alteration in areference sequence, whereby a reference sequence is any of the sequenceslisted in the “Table of Sequences and SEQ ID Numbers” (sequencelisting), i.e. SEQ ID NO:1 to SEQ ID NO:218. Thus, the term “sequencevariant” includes nucleotide sequence variants and amino acid sequencevariants. Of note, the sequence variants referred to herein are inparticular functional sequence variants, i.e. sequence variantsmaintaining the biological function of, for example, the antibody. Inthe context of the present invention such a maintained biologicalfunction is preferably the binding of the antibody to the RABV (andnon-RABV) G protein.

Sequence identity is usually calculated with regard to the full lengthof the reference sequence (i.e. the sequence recited in theapplication). Percentage identity, as referred to herein, can bedetermined, for example, using BLAST using the default parametersspecified by the NCBI (the National Center for BiotechnologyInformation; ncbi.nlm.nih.gov/) [Blosum 62 matrix; gap open penalty=11and gap extension penalty=1].

As used herein, a “nucleotide sequence variant” has an altered sequencein which one or more of the nucleotides in the reference sequence isdeleted, or substituted, or one or more nucleotides are inserted intothe sequence of the reference nucleotide sequence. Nucleotides arereferred to herein by the standard one-letter designation (A, C, G, orT). Due to the degeneracy of the genetic code, a “nucleotide sequencevariant” can either result in a change in the respective reference aminoacid sequence, i.e. in an “amino acid sequence variant” or not.Preferred sequence variants are such nucleotide sequence variants, whichdo not result in amino acid sequence variants (silent mutations), butother non-silent mutations are within the scope as well, in particularmutant nucleotide sequences, which result in an amino acid sequence,which is at least 80%, preferably at least 90%, more preferably at least95% sequence identical to the reference sequence.

An “amino acid sequence variant” has an altered sequence in which one ormore of the amino acids in the reference sequence is deleted orsubstituted, or one or more amino acids are inserted into the sequenceof the reference amino acid sequence. As a result of the alterations,the amino acid sequence variant has an amino acid sequence which is atleast 80% identical to the reference sequence, preferably, at least 90%identical, more preferably at least 95% identical, most preferably atleast 99% identical to the reference sequence. Variant sequences whichare at least 90% identical have no more than 10 alterations, i.e. anycombination of deletions, insertions or substitutions, per 100 aminoacids of the reference sequence.

While it is possible to have non-conservative amino acid substitutions,it is preferred that the substitutions be conservative amino acidsubstitutions, in which the substituted amino acid has similarstructural or chemical properties with the corresponding amino acid inthe reference sequence. By way of example, conservative amino acidsubstitutions involve substitution of one aliphatic or hydrophobic aminoacids, e.g. alanine, valine, leucine and isoleucine, with another;substitution of one hydoxyl-containing amino acid, e.g. serine andthreonine, with another; substitution of one acidic residue, e.g.glutamic acid or aspartic acid, with another; replacement of oneamide-containing residue, e.g. asparagine and glutamine, with another;replacement of one aromatic residue, e.g. phenylalanine and tyrosine,with another; replacement of one basic residue, e.g. lysine, arginineand histidine, with another; and replacement of one small amino acid,e.g., alanine, serine, threonine, methionine, and glycine, with another.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includethe fusion to the N- or C-terminus of an amino acid sequence to areporter molecule or an enzyme.

Importantly, the alterations in the sequence variants do not abolish thefunctionality of the respective reference sequence, in the present case,e.g., the functionality of a sequence of an antibody, or antigen bindingfragment thereof, to bind to the same epitope and/or to sufficientlyneutralize infection of RABV and non-RABV lyssaviruses. Guidance indetermining which nucleotides and amino acid residues, respectively, maybe substituted, inserted or deleted without abolishing suchfunctionality are found by using computer programs well known in theart.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

It is to be understood that this invention is not limited to theparticular methodology, protocols and reagents described herein as thesemay vary. It is also to be understood that the terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

The invention is based, amongst other findings, on the discovery andisolation of antibodies that are highly potent in neutralizing RABV andnon-RABV lyssaviruses, as well as of antigenic sites and epitopes towhich the antibodies of the invention bind. Such antibodies aredesirable, as only small quantities of the antibodies are required inorder to neutralize RABV and non-RABV lyssavirus infection by one singleantibody. Each single antibody according to the present invention ishighly effective in preventing as well as treating or attenuating RABVand non-RABV lyssavirus infection. Thereby, costs of production ofmedicaments comprising the antibodies for the treatment of RABV andnon-RABV lyssavirus infection are reduced. Moreover, the antibodiesaccording to the present invention neutralize not only RABV, but alsonon-RABV lyssaviruses, which also cause rabies, in a potent andeffective way. In respect to RABV the antibodies according to thepresent invention recognize broad variations, which occur naturally inthe epitopes, thus avoiding resistant RABV strains. In addition, thepeptidic antigenic sites or immunogenic polypeptides comprising epitopesrecognized by the antibodies of the invention may be a component of avaccine or a combination therapy capable of inducing protection againstRABV and non-RABV lyssavirus (reflecting active immunization).

In a first aspect, the present invention provides an isolated antibody,antibody variants and antigen binding fragments thereof, that neutralizelyssavirus infection by (i) RABV and (ii) at least 50% of non-RABVlyssaviruses selected from the group consisting of DUVV, EBLV-1, EBLV-2,ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV and WCBV, with an IC₅₀ ofless than 10000 ng/ml.

Thereby, “with an IC₅₀ of less than 10000 ng/ml” means that each speciesneutralized by an inventive antibody is inhibited by the above IC₅₀value.

“At least 50% of non-RABV lyssaviruses selected from the groupconsisting of DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK,SHIV, BBLV and WCBV” refers to at least 50% of the species Duvenhagevirus (DUVV), European bat lyssavirus type 1 (EBLV-1) and type 2(EBLV-2), Australian bat lyssavirus (ABLV), Irkut virus (IRKV), Khujandvirus (KHUV), Aravan virus (ARAV), Lagos bat virus (LBV), Mokola virus(MOKV), Shimoni bat virus (SHIV or SHIBV), Bokeloh bat lyssavirus(BBLV), and West Caucasian bat virus (WCBV), i.e. at least 6 speciesamong the above mentioned 12 species.

Preferably, the isolated antibody, antibody variants and antigen bindingfragments thereof, neutralize lyssavirus infection by (i) RABV and (ii)at least 50% of non-RABV lyssaviruses selected from the group consistingof DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV,WCBV and IKOV, with an IC₅₀ of less than 10000 ng/ml.

“At least 50% of non-RABV lyssaviruses selected from the groupconsisting of DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK,SHIV, BBLV, WCBV and IKOV” refers to at least 50% of the speciesDuvenhage virus (DUVV), European bat lyssavirus type 1 (EBLV-1) and type2 (EBLV-2), Australian bat lyssavirus (ABLV), Irkut virus (IRKV),Khujand virus (KHUV), Aravan virus (ARAV), Lagos bat virus (LBV), Mokolavirus (MOKV), Shimoni bat virus (SHIBV), Bokeloh bat lyssavirus (BBLV),West Caucasian bat virus (WCBV) and Ikoma lyssavirus (IKOV) i.e. atleast 7 species among the above mentioned 13 species.

Each single lyssavirus species is considered as being neutralized withan IC₅₀ of less than 10000 ng/ml, whenever at least one isolate of anysuch lyssavirus species is neutralized with an IC₅₀ of less than 10000ng/ml. Preferably, at least two isolates of any such lyssavirus speciesare neutralized with an IC₅₀ of less than 10000 ng/ml.

Preferably, the IC₅₀ of less than 10000 ng/ml is achieved withinfectious viruses, i.e. in particular not with pseudotyped viruses.

Thus, the antibodies, antibody variants and antigen binding fragmentsthereof, according to the present invention are able to neutralize abroad spectrum of lyssaviruses.

Preferably, the isolated antibody, antibody variants and antigen bindingfragments thereof neutralizes lyssavirus infection of at least 55%, morepreferably at least 60%, even more preferably at least 65%, mostpreferably at least 68% and particularly preferably at least 70% of thenon-RABV lyssaviruses selected from the group consisting of DUVV,EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV and WCBVwith an IC₅₀ below 10000 ng/ml.

More preferably, the isolated antibody, antibody variants and antigenbinding fragments thereof neutralizes lyssavirus infection of at least55%, more preferably at least 60%, even more preferably at least 65%,most preferably at least 68% and particularly preferably at least 70% ofthe non-RABV lyssaviruses selected from the group consisting of DUVV,EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV, WCBV andIKOV with an IC₅₀ below 10000 ng/ml.

Moreover, the present invention also provides an isolated antibody,antibody variants and antigen binding fragments thereof, that neutralizelyssavirus infection by (i) RABV and (ii) at least 50% of all isolatesof non-RABV lyssaviruses selected from the group consisting of DUVV,EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV and WCBV,with an IC₅₀ of less than 10000 ng/ml. Preferably, the isolatedantibody, antibody variants and antigen binding fragments thereof,neutralize lyssavirus infection by (i) RABV and (ii) at least 50% of allisolates of non-RABV lyssaviruses selected from the group consisting ofDUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV, WCBVand IKOV, with an IC₅₀ of less than 10000 ng/ml.

Thereby, “at least 50% of isolates of non-RABV lyssaviruses selectedfrom the group consisting of DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV,ARAV, LBV, MOK, SHIV, BBLV and WCBV” refers to all isolates of the above12 species considered and “at least 50% of all isolates of non-RABVlyssaviruses selected from the group consisting of DUVV, EBLV-1, EBLV-2,ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV, WCBV and IKOV” refers toall isolates of the above 13 species considered (i.e. all isolatesconsidered represent 100% and the number of isolates neutralized with anIC₅₀ of less than 10000 ng/ml represents the respective percentage).Preferably, the following first 31 isolates are considered to reflect100% isolates (cf. Table 1, all isolates except IKOV), more preferablyall 32 isolates shown in Table 1 (including IKOV) are considered toreflect 100% isolates:

TABLE 1 non-RABV lyssavirus isolates Viral Isolate name speciesPhylogroup ABLV/Australia/bat/9810AUS-1998/ ABLV I V1039-2011 98010 ABLVI 1301 Bokeloh bat lyssavirus BBLV I 86132SA DUVV IDUW/SouthAfrica/human/ DUVV I 96132SA-1971/RS639-2012 EBLV1a/France/bat/EBLV-1 I 122938-2002/V3951 -2009 EBLV1b/France/bat/ EBLV-1 I 8918-1989EBLV2/UK/bat/RV1332-2002/ EBLV-2 I V3951-2009 94112 EBLV-2 I 02053EBLV-2 I 8619 LBV II MOK MOK II Shimoni bat Virus SHIV II West Caucasianbat Virus WCBV III Australian bat lyssavirus/ ABLV I RV634 Aravan VirusARAV I Duvenhage Virus RSA2006 DUVV I Duvenhage Virus ZIM86-RV 131 DUVVI European bat lyssavirus 1.RV20 EBLV-1 I European bat lyssavirus 1.RV9EBLV-1 I EBLV 1a/France/bat/ EBLV-1 I 122938-2002/V3951-2009*EBLV2/UK/bat/RV 1332-2002/V3951-2009* EBLV-2 I European bat lyssavirusEBLV-2 I 2.RV1787 European bat lyssavirus EBLV-2 I 2.RV628 Irkut VirusIRKV I Khujand Virus KHUV I 8619* LBV II Lagos Bat Virus NIG56-RV1 LBVII Lagos Bat Virus SA2004 LBV II Mokola Virus NIG68.RV4 MOK II MokolaVirus 98/071 RA36 MOK II Ikoma lyssavirus IKOV IV *same isolate testedas pseudovirus and infectious virus.

A more detailed description of the non-RABV lyssavirus isolates shown inTable 1 (as well as of various RABV isolates) is shown in FIG. 1 . Thisincludes—in addition to isolate name, viral species and phylogroup (asshown in Table 1)—host species, country and year of origin, lineage andthe GenBank accession number of the amino acid and/or nucleotidesequence of the glycoprotein G of that isolate, if available (cf. FIG. 1).

Accordingly, if at least 16 of the first 31 isolates specified in Table1 (i.e. all isolates except IKOV) are neutralized by the antibody,antibody variant or antigen binding fragment thereof, with an IC₅₀ ofless than 10000 ng/ml, the antibody, antibody variant or antigen bindingfragment thereof neutralizes infection of at least 50% of isolates ofnon-RABV lyssaviruses selected from the group consisting of DUVV,EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV and WCBV,with an IC₅₀ of less than 10000 ng/ml. Moreover, if at least 16 of the32 isolates specified in Table 1 are neutralized by the antibody,antibody variant or antigen binding fragment thereof, with an IC₅₀ ofless than 10000 ng/ml, the antibody, antibody variant or antigen bindingfragment thereof neutralizes infection of at least 50% of isolates ofnon-RABV lyssaviruses selected from the group consisting of DUVV,EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV, WCBV andIKOV with an IC₅₀ of less than 10000 ng/ml.

Preferably, the isolated antibody, antibody variants and antigen bindingfragments thereof neutralizes lyssavirus infection of at least 55%, morepreferably at least 60%, even more preferably at least 65%, mostpreferably at least 68% and particularly preferably at least 70% ofisolates of the non-RABV lyssaviruses selected from the group consistingof DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLVand WCBV with an IC₅₀ of less than 10000 ng/ml. More preferably, theisolated antibody, antibody variants and antigen binding fragmentsthereof neutralizes lyssavirus infection of at least 55%, morepreferably at least 60%, even more preferably at least 65%, mostpreferably at least 68% and particularly preferably at least 70% of allisolates of the non-RABV lyssaviruses selected from the group consistingof DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV,WCBV and IKOV with an IC₅₀ below 10000 ng/ml.

Preferably, the first 12 isolates mentioned in Table 1 (i.e. fromABLV/Australia/bat/98 10ALTS-1998/V/039-2011 to MOK/MOK) are tested asinfectious viruses, whereas the other isolates mentioned in Table 1(i.e. from Shimoni bat Virus/SHIV to Ikoma lyssavirus/IKOV) arepreferably tested as pseudotyped viruses. Thereby, it is preferred toconsider an isolate as neutralizing a certain virus for infectiousviruses, if the IC₅₀ is less than 10000 ng/ml and for pseudotypedviruses, if the IC₉₀ is less than 10000 ng/ml.

Thus, in a preferred embodiment the present invention provides anisolated antibody, antibody variants and antigen binding fragmentsthereof, that neutralize lyssavirus infection by (i) RABV and (ii) atleast 50% of all isolates of non-RABV lyssaviruses selected from thegroup consisting of ABLV/Australia/bat/9810A US-1998/V1039-2011/ABLV,98010/ABLV, 1301 Bokeloh bat lyssavirus/BBLV, 86132SA/DUVV,DUVV/SouthAfrica/human/96132SA-1971/RS639-2012/DUVV,EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV1b/France/bat/8918-1989/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, 94112/EBLV-2, 02053/EBLV-2,8619/LBV, MOK/MOK, Shimoni bat Virus/SHIV, West Caucasian batVirus/WCBV, Australian bat lyssavirus/RV634/ABLV, Aravan Virus/ARAV,Duvenhage Virus RSA2006/DUVV, Duvenhage Virus ZIM86-RV 131/DUVV,European bat lyssavirus 1.RV20/EBLV-1, European bat lyssavirus1RV9/EBLV-i, EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV2/UK/bat/RI/1332-2002/V3951-2009/EBLV-2, European bat lyssavirus2.RV1787/EBLV-2, European bat Lyssavirus 2.RV628/EBLV-2, IrkutVirus/IRKV, Khujand Virus/KHUV, 8619/LBV, Lagos Bat Virus NIG56-RV1/LBV,Lagos Bat Virus SA2004/LBV, Mokola Virus NIG68.RV4/MOK, Mokola Virus98/071 RA36/MOK and Ikoma lyssavirus/IKOV with an IC₅₀ of less than10000 ng/ml for ABLV/Australia/bat/98 10AUS-1998/V1039-2011/ABLV,98010/ABLV, 1301 Bokeloh bat lyssavirus/BBLV, 86132SA/DUVV,DUVV/SouthAfrica/human/96132SA-19711 RS639-2012/DUVV,EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV1b/France/bat/8918-1969/EBLV-1,EBLV2/UK/bat/RV332-2002/V3951-2009/EBLV-2, 94112/EBLV-2, 02053/EBLV-2,8619/LBV, MOK/MOK tested as infectious viruses and with an IC₉₀ of lessthan 10000 ng/ml for Shimoni bat Virus/SHIV, West Caucasian batVirus/WCBV, Australian bat lyssavirus/RV634/ABLV, Aravan Virus/ARAV,Duvenhage Virus RSA2006/DUVV, Duvenhage Virus ZIM86-RV131/DUVV, Europeanbat lyssavirus 1.RV20/EBLV-1, European bat lyssavirus 1.RV9/EBLV-1,EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, European bat lyssavirus2.RV1787/EBLV-2, European bat Lyssavirus 2.RV628/EBLV-2, IrkutVirus/IRKV, Khujand Virus/KHUV, 8619/LBV, Lagos Bat Virus NIG56-RV1/LBV,Lagos Bat Virus SA2004/LBV, Mokola Virus NIG68.RV4/MOK, Mokola Virus98/071 RA36/MOK and Ikoma lyssavirus/IKOV tested as pseudotyped viruses.

The following description of the present invention, in particular allpreferred embodiments and further aspects, relates to both, the isolatedantibody, or an antigen binding fragment thereof, that neutralizeslyssavirus infection by (i) RABV and (ii) at least 50% of non-RABVlyssaviruses selected from the group consisting of DUVV, EBLV-1, EBLV-2,ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV and WCBV, with an IC₅₀ ofless than 10000 ng/ml as described above (including the above describedpreferred embodiments thereof) as well as to the isolated antibody, oran antigen binding fragment thereof, that neutralizes lyssavirusinfection by (i) RABV and (ii) at least 50% of (all) isolates ofnon-RABV lyssaviruses selected from the group consisting of DUVV,EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV and WCBV,with an IC₅₀ of less than 10000 ng/ml as described above (including theabove described preferred embodiments thereof).

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention binds to the G protein (glycoproteinG) of RABV. More preferably the antibody, or the antigen bindingfragment thereof, according to the present invention binds also to the Gprotein (glycoprotein G) of non-RABV lyssaviruses.

The antibody, antibody variant or antigen binding fragment thereof,according to the present invention have very low IC₅₀ values. Inparticular, antibodies with IC₅₀ values of 10000 ng/ml or more areunlikely to be effective in vivo. Thus, the antibodies according to thepresent invention have a particular high or strong affinity for RABV andnon-RABV lyssaviruses and are therefore particularly suitable forcounteracting and/or at least in part preventing a RABV- and/ornon-RABV-lyssavirus-infection and/or adverse effects of a RABV- and/ornon-RABV-lyssavirus infection.

To determine the IC₅₀ value in a neutralization assay, pseudovirusesand/or infectious viruses may be used. Respective neutralization assaysare known to the person skilled in the art. However, preferably theneutralization assay according to Wright, E. et al., Vaccine 27,7178-7186, 2009, which is incorporated by reference herein, is used forassessing pseudoviruses (PV). For infectious viruses preferably the“fluorescent-antibody virus neutralization test” (FAVN) according toCliquet, F., et al., J. Immunol Methods 212, 79-87, 1998, which isincorporated by reference herein, or “the rapid fluorescent focusinhibition test” (RFFIT) according to Smith, J. S., et al., Bull. WorldHealth Organ. 48, 535-541, 1973, which is also incorporated by referenceherein, is used.

In general, a neutralization assay typically measures the loss ofinfectivity of the virus through reaction of the virus with specificantibodies. Typically, a loss of infectivity is caused by interferenceby the bound antibody with any of the virus replication steps includingbinding to target cells, entry, and/or viral release. In the following anon-limiting example of a neutralization assay is given to illustratethe principle: a given amount of a virus, e.g. 50-100 TCDID50 (50%tissue culture infective dose), and different concentrations of theantibodies are mixed under appropriate conditions, e.g. for 1 hour atroom temperature, and then inoculated into an appropriate target cellculture, e.g. Hep-2 cells or BHK-21 (baby hamster kidney 21) cells.Values may be typically provided per ml cell culture. The presence ofunneutralized virus is detected for example after a predetermined amountof time, e.g. 1, 2, 3, 4, 5, 6, or 7 days, by measuring the cytopathiceffect of the (unneutralized) virus on target cells, e.g. by using acolorimetric assay for the quantification of cellular viability, likefor instance the WST-1 reagent. The more target cells are rescued fromcell death or are measured to be viable, the more virus was neutralizedby the antibodies. The effects measured are usually dose-dependent: Thehigher the antibody titer, the more cells are rescued. Depending on theneutralizing character of the antibody, the TCID₅₀ values vary, e.g. anantibody of significant neutralizing character will require loweramounts (of the antibody) to be added (for, e.g., achieving the sameamount of “rescued” target cells in the assay, i.e. cells measured to beviable) than another antibody of less pronounced neutralizing character.

Preferably, for the antibody according to the present invention the IC₅₀value (i.e. 50% neutralization) in an infectious virus neutralizationassay as described above is less than 10000 ng/ml, i.e. regardinginfectious viruses, whereas for the same antibody the IC₉₀ value (i.e.90% neutralization) in a pseudovirus neutralization assay as describedabove is less than 10000 ng/ml, i.e. regarding pseudoviruses.

Moreover, it is preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention neutralizeslyssavirus infection by at least 70% of non-RABV phylogroup Ilyssaviruses selected from the group consisting of DUVV, EBLV-1, EBLV-2,ABLV, IRKV, KHUV, and ARAV, with an IC₅₀ of less than 10000 ng/ml.Thereby, “at least 70%” of the above mentioned species means at least 5out of the 7 species. Infection of one lyssavirus species is consideredas neutralized with an IC₅₀ of less than 10000 ng/ml, if infection of atleast one isolate of this lyssavirus species is neutralized with an IC₅₀of less than 10000 ng/ml. Preferably, the isolated antibody, antibodyvariants and antigen binding fragments thereof neutralizes lyssavirusinfection by at least 70% of non-RABV phylogroup I lyssaviruses selectedfrom the group consisting of DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV,ARAV and BBLV with an IC₅₀ of less than 10000 ng/ml. Thereby, “at least70%” of the above mentioned species means at least 6 out of the 8species. Infection of one lyssavirus species is considered asneutralized with an IC₅₀ of less than 10000 ng/ml, if infection of atleast one isolate of this lyssavirus species is neutralized with an IC₅₀of less than 10000 ng/ml.

Preferably, the isolated antibody, antibody variants and antigen bindingfragments thereof neutralizes lyssavirus infection of at least 75%, morepreferably at least 80%, even more preferably at least 82%, of thenon-RABV phylogroup I lyssaviruses selected from the group consisting ofDUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, and ARAV, with an IC₅₀ of lessthan 10000 ng/ml. More preferably, the isolated antibody, antibodyvariants and antigen binding fragments thereof neutralizes lyssavirusinfection of at least 75%, more preferably at least 80%, even morepreferably at least 82%, of the non-RABV phylogroup I lyssavirusesselected from the group consisting of DUVV, EBLV-1, EBLV-2, ABLV, IRKV,KHUV, ARAV and BBLV with an IC₅₀ of less than 10000 ng/ml.

According to another preferred embodiment, the antibody, or the antigenbinding fragment thereof, according to the present invention neutralizeslyssavirus infection by at least 70% of isolates of non-RABV phylogroupI lyssaviruses selected from the group consisting of DUVV, EBLV-1,EBLV-2, ABLV, IRKV, KHUV, and ARAV, with an IC₅₀ of less than 10000ng/ml. Thereby, “at least 70% of isolates of non-RABV lyssavirusesselected from the group consisting of DUVV, EBLV-1, EBLV-2, ABLV, IRKV,KHUV, and ARAV” refers to all isolates of the above 7 species considered(i.e. all isolates considered represent 100% and the number of isolatesneutralized with an IC₅₀ of less than 10000 ng/ml represents therespective percentage). More preferably, the antibody, or the antigenbinding fragment thereof, according to the present invention neutralizeslyssavirus infection by at least 70% of isolates of non-RABV phylogroupI lyssaviruses selected from the group consisting of DUVV, EBLV-1,EBLV-2, ABLV, IRKV, KHUV, ARAV and BBLV with an IC₅₀ of less than 10000ng/ml. Thereby, “at least 70% of isolates of non-RABV lyssavirusesselected from the group consisting of DUVV, EBLV-1, EBLV-2, ABLV, IRKV,KHUV ARAV and BBLV” refers to all isolates of the above 8 speciesconsidered (i.e. all isolates considered represent 100% and the numberof isolates neutralized with an IC₅₀ of less than 10000 ng/ml representsthe respective percentage). Preferably, the 22 isolates mentioned inTable 1 regarding phylogroup I species are considered to calculate thepercentage. Accordingly, if at least 16 out of these 22 isolatesmentioned in Table 1 regarding phylogroup I species are neutralized bythe antibody, antibody variant or antigen binding fragment thereof, withan IC₅₀ of less than 10000 ng/ml, the antibody, antibody variant orantigen binding fragment thereof neutralizes infection of at least 70%of isolates of non-RABV phylogroup I lyssaviruses selected from thegroup consisting of DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV and,preferably BBLV, with an IC₅₀ of less than 10000 ng/ml. Preferably, theisolated antibody, antibody variants and antigen binding fragmentsthereof neutralizes lyssavirus infection of at least 75%, morepreferably at least 80%, even more preferably at least 82%, of isolatesof the non-RABV phylogroup I lyssaviruses selected from the groupconsisting of DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV and,preferably, BBLV with an IC₅₀ of less than 10000 ng/ml.

It is particularly preferred that the isolated antibody, antibodyvariants and antigen binding fragments thereof, according to the presentinvention neutralizes lyssavirus infection by at least 70% of theisolates of non-RABV phylogroup I lyssaviruses selected from the groupconsisting of ABLV/Australia/bat/9810AUS-1998/V1039-201/ABLV,98010/ABLV, 1301 Bokeloh bat lyssavirus/BBLV, 86132SA/DUVV,DUVV/SouthAfrica/human/96132 SA-1971/RS639-2012/DUVV, EBLVFrance/bat/122938-2002/V3951-2009/EBLV-1,EBLV1b/France/bat/8918-1989/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, 94112/EBLV-2, 02053/EBLV-2,Australian bat lyssavirus/RV634/ABLV, Aravan Virus/ARAV, Duvenhage VirusRSA2006/DUVV, Duvenhage Virus ZIM86-RV 131/DUVV, European bat lyssavirus1.RV20/EBLV-1, European bat lyssavirus 1.RV9/EBLV-1,EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, European bat lyssavirus2.RV1787/EBLV-2 and European bat lyssavirus 2.RV628/EBLV-2, IrkutVirus/IRKV, Khujand Virus/KHUV, with an IC₅₀ of less than 10000 ng/mlfor ABLV/Australia/bat/98 10AUS-1998/V1039-2011/ABLV, 98010/ABLV, 1301Bokeloh bat lyssavirus/BBLV, 86132SA/DUVV,DUVV/SouthAfrica/human/96132SA-1971/RS639-2012/DUVV, EBLVFrance/bat/122938-2002/V3951-2009/EBLV-1,EBLV113/France/bat/8918-1989/EBLV-1,EBLV2/UK/bat/RV332-2002/V3951-2009/EBLV-2, 94412/EBLV-2 and02053/EBLV-2, tested as infectious viruses and with an IC₉₀ of less than10000 ng/ml for Australian bat lyssavirus/RV634/ABLV, Aravan Virus/ARAV,Duvenhage Virus RSA2006/DUVV, Duvenhage Virus ZIM86-RV131/DUVV, Europeanbat lyssavirus 1.RV20/EBLV-1, European bat lyssavirus 1.RV9/EBLV-1,EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, European bat lyssavirus2.RV1787/EBLV-2, European bat lyssavirus 2.RV628/EBLV-2, IrkutVirus/IRKV and Khujand Virus/KHUV tested as pseudotyped viruses.

Among the lyssaviruses, and in particular the phylogroup I lyssaviruses,it is particularly preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention neutralizesinfection of EBLV-i. In particular, the antibody, or the antigen bindingfragment thereof, according to the present invention neutralizesinfection of at least one isolate of EBLV-1, more preferably of at leasttwo, even more preferably of at least three EBLV-1 isolates, e.g. theEBLV-1 isolates mentioned in Table 1, with an IC₅₀ below 10000 ng/ml.Thereby, it is particularly preferred if the one or more EBLV-1 isolatesare neutralized in both, in a pseudovirus neutralization assay asdescribed above and in an infectious virus neutralization assay asdescribed above. Even more preferably, the IC₅₀ value in the infectiousvirus neutralization assay is below 10000 ng/ml and the IC₉₀ value inthe pseudovirus neutralization assay is below 10000 ng/ml.

Of note, none of the prior art human monoclonal antibodies CR57, CR4098and RAM neutralizes EBLV-1 (cf. FIGS. 5 and 6 ). However, rabies due toEuropean Bat Lyssavirus type 1 is present in many European countries andbats are listed as protected species across Europe. The disease is fatalin humans and has been described in Europe following a bat bite. Rabiespre-exposure vaccination and post-exposure treatment is recommended foroccupationally exposed persons and treatment of international travelersafter bat bites is also recommended (Stantic-Pavlinic M. (2005)Eurosurveillance, Volume 10, Issue ii), however, as shown in FIGS. 5 and6, HRIG hardly neutralizes EBLV-1. Therefore, there is a need for(human) antibodies neutralizing EBLV-1.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention neutralizes infection byRABV CVS-11 with an IC₉₀ of 400 ng/ml or less, more preferably with anIC₉₀ of 100 ng/ml or less, even more preferably with an IC₉₀ of 50 ng/mlor less and particularly preferably with an IC₉₀ of 1 ng/ml or less. Inother words, the concentration of the antibody, or the antigen bindingfragment thereof, according to the present invention required for 90%neutralization (IC₉₀) of the RABV isolate CVS-11 (challenge virus strain11) is 400 ng/ml or less, preferably 100 ng/ml or less, more preferably50 ng/ml or less, even more preferably 1 ng/ml or less, in particular ina pseudovirus neutralization assay as described above, and preferablyalso in a RFFIT infectious virus neutralization assay as describedabove.

In general, it is preferred that the antibody, or the antigen bindingfragment thereof, according to the present invention is a monoclonalantibody or antigen binding fragment thereof. In contrast to polyclonalantibodies, monoclonal antibodies are monospecific antibodies, i.e. theybind to a specific epitope. Therefore, unexpected binding is largelyavoided and monoclonal antibodies are considered as safer compared topolyclonal antibodies.

Preferably, the antibody according to the present invention, or theantigen binding fragment thereof, is a human antibody, a monoclonalantibody, a human monoclonal antibody, a purified antibody, a singlechain antibody, Fab, Fab′, F(ab′)2, Fv or scFv.

The antibodies of the invention may thus be human antibodies, monoclonalantibodies, human monoclonal antibodies, recombinant antibodies orpurified antibodies. The invention also provides fragments of theantibodies of the invention, particularly fragments that retain theantigen-binding activity of the antibodies. Such fragments include, butare not limited to, single chain antibodies, Fab, Fab′, F(ab′)2, Fv orscFv. Although the specification, including the claims, may, in someplaces, refer explicitly to antigen binding fragment(s), antibodyfragment(s), variant(s) and/or derivative(s) of antibodies, it isunderstood that the term “antibody” or “antibody of the invention”includes all categories of antibodies, namely, antigen bindingfragment(s), antibody fragment(s), variant(s) and derivative(s) ofantibodies.

Fragments of the antibodies of the invention can be obtained from theantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, fragments of the antibodies can be obtained bycloning and expression of part of the sequences of the heavy or lightchains. Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fvfragments. The invention also encompasses single-chain Fv fragments(scFv) derived from the heavy and light chains of an antibody of theinvention. For example, the invention includes a scFv comprising theCDRs from an antibody of the invention. Also included are heavy or lightchain monomers and dimers, single domain heavy chain antibodies, singledomain light chain antibodies, as well as single chain antibodies, e.g.,single chain Fv in which the heavy and light chain variable domains arejoined by a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalentinteractions and be contained in a variety of structures as describedabove. For instance, scFv molecules may be synthesized to create atrivalent “triabody” or a tetravalent “tetrabody.” The scFv moleculesmay include a domain of the Fc region resulting in bivalent minibodies.In addition, the sequences of the invention may be a component ofmultispecific molecules in which the sequences of the invention targetthe epitopes of the invention and other regions of the molecule bind toother targets. Exemplary molecules include, but are not limited to,bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies(Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

In general, the antibody according to the present invention, or theantigen binding fragment thereof, preferably comprises (at least) threeCDRs on the heavy chain and (at least) three CDRs on the light chain. Ingeneral, complementarity determining regions (CDRs) are thehypervariable regions present in heavy chain variable domains and lightchain variable domains. Typically, the CDRs of a heavy chain and theconnected light chain of an antibody together form the antigen receptor.Usually, the three CDRs (CDR1, CDR2, and CDR3) are arrangednon-consecutively in the variable domain. Since antigen receptors aretypically composed of two variable domains (on two different polypeptidechains, i.e. heavy and light chain), there are six CDRs for each antigenreceptor (heavy chain: CDRH1, CDRH2, and CDRH3; light chain: CDRL1,CDRL2, and CDRL3). A single antibody molecule usually has two antigenreceptors and therefore contains twelve CDRs. The CDRs on the heavyand/or light chain may be separated by framework regions, whereby aframework region (FR) is a region in the variable domain which is less“variable” than the CDR. For example, a chain (or each chain,respectively) may be composed of four framework regions, separated bythree CDR.

Preferably, the CDRs, in particular CDRH3, of the antibody according tothe present invention are derived from an antibody developed in a human.In particular, the CDRs, in particular the CDRH3, of the antibodyaccording to the present invention are of human origin or functionalsequence variants thereof.

The sequences of the heavy chains and light chains of several antibodiesof the invention, each comprising three CDRs on the heavy chain andthree CDRs on the light chain have been determined. The position of theCDR amino acids are defined according to the IMGT numbering system(IMGT: imgt.org/; cf. Lefranc, M.-P. et al. (2009) Nucleic Acids Res.37, D1006-D1012). The sequences of the CDRs, heavy chains, light chainsas well as the sequences of the nucleic acid molecules encoding theCDRs, heavy chains, light chains of the antibodies of the invention,i.e. of several antibodies according to the invention, are disclosed inthe sequence listing. The CDRs of the antibody heavy chains are alsoreferred to as CDRH1, CDRH2 and CDRH3, respectively. Similarly, the CDRsof the antibody light chains are also referred to as CDRL1, CDRL2 andCDRL3, respectively.

Preferably, the antibody according to the present invention, or theantigen binding fragment thereof, comprises a heavy chain comprisingCDRH1, CDRH2 and CDRH3 and a light chain comprising CDRL1, CDRL2 andCDRL3, wherein the heavy chain CDRH3 comprises an amino acid sequencethat is at least 80%, preferably at least 90%, identical to SEQ ID NOs:3, 21, 39, 57, 77, 95, 113, 131, 149, 167, 185, or 203, whereby aminoacid sequences that are at least 80%, preferably at least 90%, identicalto SEQ ID NOs: 95 or 167 are particularly preferred. More preferably,the heavy chain CDRH3 of the antibody, or of the antigen bindingfragment thereof, comprises the amino acid sequence of SEQ ID NOs: 3,21, 39, 57, 77, 95, 113, 131, 149, 167, 185, or 203, more preferably ofSEQ ID NOs: 95 or 167. In more general terms, the present invention alsocomprises an antibody, or an antigen binding fragment thereof,comprising a heavy chain comprising CDRH1, CDRH2 and CDRH3 and a lightchain comprising CDRL1, CDRL2 and CDRL3, wherein the heavy chain CDRH3comprises an amino acid sequence variant to SEQ ID NOs: 3, 21, 39, 57,77, 95, 113, 131, 149, 167, 185, or 203, whereby amino acid sequencevariants to SEQ ID NOs: 95 or 167 are particularly preferred. Morepreferably the heavy chain comprises at least two CDRH3 with one heavychain CDRH3 comprising an amino acid sequence that is at least 80%,preferably at least 90%, identical to SEQ ID NO: 95 and one heavy chainCDRH3 comprising an amino acid sequence that is at least 80%, preferablyat least 90%, identical to SEQ ID NO: 167. The sequence variantsreferred to above are in particular functional sequence variants, e.g.wherein the binding of the antibody to the RABV (and non-RABV) G protein(glycoprotein G) is maintained.

It is also preferred that, the isolated antibody of the invention, orthe antigen binding fragment thereof, comprises a heavy chain comprisingCDR1, CDR2 and CDR3 and a light chain comprising CDR1, CDR2 and CDR3,wherein the heavy chain CDR3 comprises an amino acid sequence that is atleast 90%, for example, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NOs: 3, 21, 39, 57, 77, 95, 113, 131, 149,167, 185, or 203, preferably to SEQ ID NOs: 95 or 167. More preferablythe heavy chain comprises at least two CDRH3 with one heavy chain CDRH3comprising an amino acid sequence of SEQ ID NO: 95 and one heavy chainCDRH3 comprising an amino acid sequence of SEQ ID NO: 167. The sequencevariants referred to above are in particular functional sequencevariants, e.g. wherein the binding of the antibody to the RABV (andnon-RABV) G protein (glycoprotein G) is maintained.

Table 2 provides the SEQ ID numbers for the amino acid sequences of thesix CDRs of the heavy and light chains, respectively, of exemplaryantibodies of the invention.

TABLE 2 SEQ ID Numbers for CDR Polypeptides of exemplary antibodies ofthe invention. SEQ ID NOs. for CDR Polypeptides CDRH1 CDRH2 CDRH3 CDRL1CDRL2 CDRL3 RVA122 1 2 3 4 5/6 7 RVA144 19 20 21 22 23/24 25 RVB185 3738 39 40 41/42 43 RVB492 55 56 57 58 59/60 61 RVC3 75 76 77 78 79/80 81RVC20 93 94 95 96 97/98 99 RVC21 111 112 113 114 115/116 117 RVC38 129130 131 132 133/134 135 RVC44 147 148 149 150 151/152 153 RVC58 165 166167 168 169/170 171 RVC68 183 184 185 186 187/188 189 RVC111 201 202 203204 205/206 207

Variant antibodies are also included within the scope of the invention.Thus, variants of the sequences recited in the application are alsoincluded within the scope of the invention. The variants referred toherein are in particular functional, e.g. wherein the binding of theantibody to the RABV (and non-RABV) G protein (glycoprotein G) ismaintained. Such variants include natural variants generated by somaticmutation in vivo during the immune response or in vitro upon culture ofimmortalized B cell clones. Alternatively, variants may arise due to thedegeneracy of the genetic code or may be produced due to errors intranscription or translation.

Further variants of the antibody sequences having improved affinityand/or potency may be obtained using methods known in the art and areincluded within the scope of the invention. For example, amino acidsubstitutions may be used to obtain antibodies with further improvedaffinity. Alternatively, codon optimization of the nucleotide sequencemay be used to improve the efficiency of translation in expressionsystems for the production of the antibody. Further, polynucleotidescomprising a sequence optimized for antibody specificity or neutralizingactivity by the application of a directed evolution method to any of thenucleic acid sequences of the invention are also within the scope of theinvention.

Preferably, variant antibody sequences may share 70% or more (i.e. 75%,80%, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more) amino acidsequence identity with the sequences recited in the application. Suchvariants usually have a greater homology to the sequences listed hereinin the CDRs of the heavy chain variable region (V_(H)) and light chainvariable region (V_(L)) than in the framework region. As is known to oneof skill in the art, mutations are more tolerated, i.e., limited or noloss of function (e.g., specificity or neutralization ability) in theframework regions than in the CDRs.

The invention thus comprises an antibody, or an antigen binding fragmentthereof, wherein the variation from the sequences provided herein ispreferably in the framework region(s) of the antibody or in the nucleicacid residues that encode the framework region(s) of the antibody.

In the present invention, such (variant) antibodies are preferred, inwhich the number of somatic mutations is reduced (i.e. “germlined”antibodies: reverted back to the “germline” configuration). Germlinesequences of antibodies may be determined, for example, with referenceto IMGT database (e.g., according to the IMGT VDJ and VJ assignments andrearrangement interpretation: imgt.org/; cf. Lefranc, M.-P. et al.(2009) Nucleic Acids Res. 37, D1006-D1012) and “germlined” antibodyvariants may be produced, for example, by gene synthesis or bysite-directed mutagenesis. A low level of somatic mutations reduces thepotential risk of antibody immunogenicity. Preferably, the number ofsomatic mutations is reduced in the framework regions (FR) (i.e.“framework regions germlined” antibodies, also referred to herein asFR-GL variants). (Variant) antibodies, or an antigen binding fragmentthereof, and FR-GL variants, respectively, without any somatic mutationsin the framework regions (FR) are more preferred. Particularly preferredare such (variant) antibodies, or an antigen binding fragment thereof,and FR-GL variants, respectively, with as few somatic mutations aspossible, whereby on the other hand the neutralizing activity is notimpaired (as compared to the reference antibody/fragment containing(more) somatic mutations). Such antibodies are on the one hand notimpaired in their neutralizing activities, thus showing a very highpotency and breadth. On the other hand, a potential risk of antibodyimmunogenicity is significantly reduced.

In a preferred embodiment, the isolated antibody or antibody fragment ofthe invention comprises at least one CDR with a sequence that has atleast 95% sequence identity to any one of SEQ ID NOs: 1-7, 19-25, 37-43,55-61, 75-81, 93-99, 111-117, 129-135, 147-153, 165-171, 183-189, or201-207. The amino acid sequences of the heavy and light chain variableregions of the antibodies of the invention as well as the nucleic acidsequences that encode them are provided in the Table of Sequences andSEQ ID Numbers below. The amino acid residues corresponding to the sixCDRs and the nucleic acid residues that encode them are highlighted inbold text.

Preferably, an isolated antibody, or antigen binding fragment thereof,according to the present invention comprises more than one CDR with asequence that has at least 95% sequence identity to any one of SEQ IDNOs: 1-7, 19-25, 37-43, 55-61, 75-81, 93-99, 111-117, 129-135, 147-153,165-171, 183-189, and 201-207.

Preferably, the antibody, or antigen binding fragment thereof, comprisestwo CDRs with a sequence that has at least 95% sequence identity to anyone of SEQ ID NOs: 1-7, 19-25, 37-43, 55-61, 75-81, 93-99, 111-117,129-135, 147-153, 165-171, 183-189, or 201-207. Thereby it is preferredthat the antibody, or antigen binding fragment thereof, comprises (i) aCDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs:1, 19, 37, 55, 75, 93, 111, 129, 147, 165, 183 or 201 and a CDRL1 thathas at least 95% sequence identity to any one of SEQ ID NOs: 4, 22, 40,58, 78, 96, 114, 132, 150, 168, 186, or 204; (ii) a CDRH2 that has atleast 95% sequence identity to any one of SEQ ID NOs: 2, 20, 38, 56, 76,94, 112, 130, 148, 166, 184, or 202, and a CDRL2 that has at least 95%sequence identity to any one of SEQ ID NOs: 5, 6, 23, 24, 41, 42, 59,60, 79, 80, 97, 98, 115, 116, 133, 134, 151, 152, 169, 170, 187, 188,205, or 206; or (iii) a CDRH3 that has at least 95% sequence identity toany one of SEQ ID NOs: 3, 21, 39, 57, 77, 95, 113, 131, 149, 167, 185,or 203, and a CDRL3 that has at least 95% sequence identity to any oneof SEQ ID NOs: 7, 25, 43, 61, 81, 99, 117, 135, 153, 171, 189, or 201.

Preferably, the antibody, or antigen binding fragment thereof, comprisesthree CDRs with a sequence that has at least 95% sequence identity toany one of SEQ ID NOs: 1-7, 19-25, 37-43, 55-61, 75-81, 93-99, 111-117,129-135, 147-153, 165-171, 183-189, and 201-207. Thereby it is preferredthat the antibody, or antigen binding fragment thereof, comprises (i) aCDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs:1, 19, 37, 55, 75, 93, 111, 129, 147, 165, 183 or 201, a CDRH2 that hasat least 95% sequence identity to any one of SEQ ID NOs: 2, 20, 38, 56,76, 94, 112, 130, 148, 166, 184, or 202, and a CDRH3 that has at least95% sequence identity to any one of SEQ ID NOs: 3, 21, 39, 57, 77, 95,113, 131, 149, 167, 185, or 203; or (ii) a CDRL1 that has at least 95%sequence identity to any one of SEQ ID NOs: 4, 22, 40, 58, 78, 96, 114,132, 150, 168, 186, or 204, a CDRL2 that has at least 95% sequenceidentity to any one of SEQ ID NOs: 5, 6, 23, 24, 41, 42, 59, 60, 79, 80,97, 98, 115, 116, 133, 134, 151, 152, 169, 170, 187, 188, 205, or 206,and a CDRL3 that has at least 95% sequence identity to any one of SEQ IDNOs: 7, 25, 43, 61, 81, 99, 117, 135, 153, 171, 189, or 201.

Preferably, the antibody, or antigen binding fragment thereof, comprisesfour CDRs with a sequence that has at least 95% sequence identity to anyone of SEQ ID NOs: 1-7, 19-25, 37-43, 55-61, 75-81, 93-99, 111-117,129-135, 147-153, 165-171, 183-189, and 201-207. Thereby it is preferredthat the antibody, or antigen binding fragment thereof, comprises (i) aCDRH1 that has at least 95% sequence identity to any one of SEQ ID NOs:1, 19, 37, 55, 75, 93, 111, 129, 147, 165, 183 or 201, a CDRH2 that hasat least 95% sequence identity to any one of SEQ ID NOs: 2, 20, 38, 56,76, 94, 112, 130, 148, 166, 184, or 202, a CDRH3 that has at least 95%sequence identity to any one of SEQ ID NOs: 3, 21, 39, 57, 77, 95, 113,131, 149, 167, 185, or 203, and a CDRL that has at least 95% sequenceidentity to any one of SEQ ID NOs: 4-6, 20-22, 36-38, or 52-54; (ii) aCDRL1 that has at least 95% sequence identity to any one of SEQ ID NOs:4, 22, 40, 58, 78, 96, 114, 132, 150, 168, 186, or 204, a CDRL2 that hasat least 95% sequence identity to any one of SEQ ID NOs: 5, 6, 23, 24,41, 42, 59, 60, 79, 80, 97, 98, 115, 116, 133, 134, 151, 152, 169, 170,187, 188, 205, or 206, a CDRL3 that has at least 95% sequence identityto any one of SEQ ID NOs: 7, 25, 43, 61, 81, 99, 117, 135, 153, 171,189, or 201, and a CDRH that has at least 95% sequence identity to anyone of SEQ ID NOs: 1-3, 19-21, 37-39, 55-57, 75-77, 93-95, 111-113,129-131, 147-149, 165-167, 183-185 or 201-203, whereby a CDRH3 that hasat least 95% sequence identity to any one of SEQ ID NOs: 3, 21, 39, 57,77, 95, 113, 131, 149, 167, 185, or 203 is particularly preferred; (iii)a CDRH1 that has at least 95% sequence identity to any one of SEQ IDNOs: 1, 19, 37, 55, 75, 93, 111, 129, 147, 165, 183 or 201, a CDRL1 thathas at least 95% sequence identity to any one of SEQ ID NOs: 4, 22, 40,58, 78, 96, 114, 132, 150, 168, 186, or 204, a CDRH2 that has at least95% sequence identity to any one of SEQ ID NOs: 2, 20, 38, 56, 76, 94,112, 130, 148, 166, 184, or 202, and a CDRL2 that has at least 95%sequence identity to any one of SEQ ID NOs: 5, 6, 23, 24, 41, 42, 59,60, 79, 80, 97, 98, 115, 116, 133, 134, 151, 152, 169, 170, 187, 188,205, or 206; (iv) a CDRH1 that has at least 95% sequence identity to anyone of SEQ ID NOs: 1, 19, 37, 55, 75, 93, 111, 129, 147, 165, 183 or201, a CDRL1 that has at least 95% sequence identity to any one of SEQID NOs: 4, 22, 40, 58, 78, 96, 114, 132, 150, 168, 186, or 204, a CDRH3that has at least 95% sequence identity to any one of SEQ ID NOs: 3, 21,39, 57, 77, 95, 113, 131, 149, 167, 185, or 203, and a CDRL3 that has atleast 95% sequence identity to any one of SEQ ID NOs: 7, 25, 43, 61, 81,99, 117, 135, 153, 171, 189, or 201; or (v) a CDRH2 that has at least95% sequence identity to any one of SEQ ID NOs: 2, 20, 38, 56, 76, 94,112, 130, 148, 166, 184, or 202, a CDRL2 that has at least 95% sequenceidentity to any one of SEQ ID NOs: 5, 6, 23, 24, 41, 42, 59, 60, 79, 80,97, 98, 115, 116, 133, 134, 151, 152, 169, 170, 187, 188, 205, or 206, aCDRH3 that has at least 95% sequence identity to any one of SEQ ID NOs:3, 21, 39, 57, 77, 95, 113, 131, 149, 167, 185, or 203, and a CDRL3 thathas at least 95% sequence identity to any one of SEQ ID NOs: 7, 25, 43,61, 81, 99, 117, 135, 153, 171, 189, or 201.

Preferably, the antibody, or antigen binding fragment thereof, comprisesfive CDRs with a sequence that has at least 95% sequence identity to anyone of SEQ ID NOs: 1-7, 19-25, 37-43, 55-61, 75-81, 93-99, 111-117,129-135, 147-153, 165-171, 183-189, and 201-207. Thereby it is preferredthat the antibody, or antigen binding fragment thereof, comprises fiveCDRs selected from the group of a CDRH1 that has at least 95% sequenceidentity to any one of SEQ ID NOs: 1, 19, 37, 55, 75, 93, 111, 129, 147,165, 183 or 201, a CDRH2 that has at least 95% sequence identity to anyone of SEQ ID NOs: 2, 20, 38, 56, 76, 94, 112, 130, 148, 166, 184, or202, a CDRH3 that has at least 95% sequence identity to any one of SEQID NOs: 3, 21, 39, 57, 77, 95, 113, 131, 149, 167, 185, or 203, a CDRL1that has at least 95% sequence identity to any one of SEQ ID NOs: 4, 22,40, 58, 78, 96, 114, 132, 150, 168, 186, or 204, a CDRL2 that has atleast 95% sequence identity to any one of SEQ ID NOs: 5, 6, 23, 24, 41,42, 59, 60, 79, 80, 97, 98, 115, 116, 133, 134, 151, 152, 169, 170, 187,188, 205, or 206, and a CDRL3 that has at least 95% sequence identity toany one of SEQ ID NOs: 7, 25, 43, 61, 81, 99, 117, 135, 153, 171, 189,or 201.

Preferably, the antibody, or antigen binding fragment thereof, comprisessix CDRs with a sequence that has at least 95% sequence identity to anyone of SEQ ID NOs: 1-7, 19-25, 37-43, 55-61, 75-81, 93-99, 111-117,129-135, 147-153, 165-171, 183-189, and 201-207. Thereby it is preferredthat the antibody, or antigen binding fragment thereof, comprises sixCDRs selected from the group of a CDRH1 that has at least 95% sequenceidentity to any one of SEQ ID NOs: 1, 19, 37, 55, 75, 93, 111, 129, 147,165, 183 or 201, a CDRH2 that has at least 95% sequence identity to anyone of SEQ ID NOs: 2, 20, 38, 56, 76, 94, 112, 130, 148, 166, 184, or202, a CDRH3 that has at least 95% sequence identity to any one of SEQID NOs: 3, 21, 39, 57, 77, 95, 113, 131, 149, 167, 185, or 203, a CDRL1that has at least 95% sequence identity to any one of SEQ ID NOs: 4, 22,40, 58, 78, 96, 114, 132, 150, 168, 186, or 204, a CDRL2 that has atleast 95% sequence identity to any one of SEQ ID NOs: 5, 6, 23, 24, 41,42, 59, 60, 79, 80, 97, 98, 115, 116, 133, 134, 151, 152, 169, 170, 187,188, 205, or 206, and a CDRL3 that has at least 95% sequence identity toany one of SEQ ID NOs: 7, 25, 43, 61, 81, 99, 117, 135, 153, 171, 189,or 201. More preferably, the antibody, or antigen binding fragmentthereof, comprises: (i) heavy chain CDRH1, CDRH2, and CDRH3 amino acidsequences and light chain CDRL1, CDRL2, and CDRL3 amino acid sequencesthat are at least 80%, preferably at least 90%, identical to the aminoacid sequences of SEQ ID NOs: 1-5 and 7 or to the amino acid sequencesof SEQ ID NOs: 1-4 and 6-7, respectively; (ii) heavy chain CDRH1, CDRH2,and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3amino acid sequences that are at least 80%, preferably at least 90%,identical to the amino acid sequences of SEQ ID NOs: 19-23 and 25 or tothe amino acid sequences of SEQ ID NOs: 19-22 and 24-25, respectively;(iii) heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and lightchain CDRL1, CDRL2, and CDRL3 amino acid sequences that are at least80%, preferably at least 90%, identical to the amino acid sequences ofSEQ ID NOs: 37-41 and 43 or to the amino acid sequences of SEQ ID NOs:37-40 and 42-43, respectively; (iv) heavy chain CDRH1, CDRH2, and CDRH3amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acidsequences that are at least 80%, preferably at least 90%, identical tothe amino acid sequences of SEQ ID NOs: 55-59 and 61 or to the aminoacid sequences of SEQ ID NOs: 55-58 and 60-61, respectively; (v) heavychain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 75-79 and 81 or to the amino acid sequences of SEQ ID NOs: 75-78and 80-81, respectively; (vi) heavy chain CDRH1, CDRH2, and CDRH3 aminoacid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acidsequences that are at least 80%, preferably at least 90%, identical tothe amino acid sequences of SEQ ID NOs: 93-97 and 99 or to the aminoacid sequences of SEQ ID NOs: 93-96 and 98-99, respectively; (vii) heavychain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 111-115 and 117 or to the amino acid sequences of SEQ ID NOs:111-114 and 116-117, respectively; (viii) heavy chain CDRH1, CDRH2, andCDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences that are at least 80%, preferably at least 90%, identicalto the amino acid sequences of SEQ ID NOs: 129-133 and 135 or to theamino acid sequences of SEQ ID NOs: 129-132 and 134-135, respectively;(ix) heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and lightchain CDRL1, CDRL2, and CDRL3 amino acid sequences that are at least80%, preferably at least 90%, identical to the amino acid sequences ofSEQ ID NOs: 147-151 and 153 or to the amino acid sequences of SEQ IDNOs: 147-150 and 152-153, respectively; (x) heavy chain CDRH1, CDRH2,and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3amino acid sequences that are at least 80%, preferably at least 90%,identical to the amino acid sequences of SEQ ID NOs: 165-169 and 171 orto the amino acid sequences of SEQ ID NOs: 165-168 and 170-171,respectively; (xi) heavy chain CDRH1, CDRH2, and CDRH3 amino acidsequences and light chain CDRL1, CDRL2, and CDRL3 amino acid sequencesthat are at least 80%, preferably at least 90%, identical to the aminoacid sequences of SEQ ID NOs: 183-187 and 189 or to the amino acidsequences of SEQ ID NOs: 183-186 and 188-189, respectively; or (xii)heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 201-205 and 207 or to the amino acid sequences of SEQ ID NOs:201-204 and 206-207, respectively.

In a particularly preferred embodiment the isolated antibody or antigenbinding fragment thereof, according to the invention comprises heavychain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 93-97 and 99 or to the amino acid sequences of SEQ ID NOs: 93-96and 98-99, respectively.

In another particularly preferred embodiment the isolated antibody orantigen binding fragment thereof, according to the invention comprisesheavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 165-169 and 171 or to the amino acid sequences of SEQ ID NOs:165-168 and 170-171, respectively.

Even more preferably, the isolated antibody or antigen binding fragmentthereof, according to the invention comprises: (i) heavy chain CDRH1,CDRH2, and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, andCDRL3 amino acid sequences as set forth in SEQ ID NOs: 1-5 and 7 or inSEQ ID NOs: 1-4 and 6-7, respectively; (ii) heavy chain CDRH1, CDRH2,and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3amino acid sequences as set forth in SEQ ID NOs: 19-23 and 25 or in SEQID NOs: 19-22 and 24-25, respectively; (iii) heavy chain CDRH1, CDRH2,and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3amino acid sequences as set forth in SEQ ID NOs: 37-41 and 43 or in SEQID NOs: 37-40 and 42-43, respectively; (iv) heavy chain CDRH1, CDRH2,and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3amino acid sequences as set forth in SEQ ID NOs: 55-59 and 61 or in SEQID NOs: 55-58 and 60-61, respectively; (v) heavy chain CDRH1, CDRH2, andCDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences as set forth in SEQ ID NOs: 75-79 and 81 or in SEQ IDNOs: 75-78 and 80-81, respectively; (vi) heavy chain CDRH1, CDRH2, andCDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences as set forth in SEQ ID NOs: 93-97 and 99 or in SEQ IDNOs: 93-96 and 98-99, respectively; (vii) heavy chain CDRH1, CDRH2, andCDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences as set forth in SEQ ID NOs: 111-115 and 117 or in SEQ IDNOs: 111-114 and 116-117, respectively; (viii) heavy chain CDRH1, CDRH2,and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3amino acid sequences as set forth in SEQ ID NOs: 129-133 and 135 or inSEQ ID NOs: 129-132 and 134-135, respectively; (ix) heavy chain CDRH1,CDRH2, and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, andCDRL3 amino acid sequences as set forth in SEQ ID NOs: 147-151 and 153or in SEQ ID NOs: 147-150 and 152-153, respectively; (x) heavy chainCDRH1, CDRH2, and CDRH3 amino acid sequences and light chain CDRL1,CDRL2, and CDRL3 amino acid sequences as set forth in SEQ ID NOs:165-169 and 171 or in SEQ ID NOs: 165-168 and 170-171, respectively;(xi) heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and lightchain CDRL1, CDRL2, and CDRL3 amino acid sequences as set forth in SEQID NOs: 183-187 and 189 or in SEQ ID NOs: 183-186 and 188-189,respectively; or (xii) heavy chain CDRH1, CDRH2, and CDRH3 amino acidsequences and light chain CDRL1, CDRL2, and CDRL3 amino acid sequencesas set forth in SEQ ID NOs: 201-205 and 207 or in SEQ ID NOs: 201-204and 206-207, respectively.

Among the embodiments described above of the antibody, or antigenbinding fragment thereof, of the invention having at least one CDR, i.e.one, two, three, four, five six CDRs as described above, such anembodiment of the antibody, or antigen binding fragment thereof, ispreferred, which comprises a CDRH3 that has at least 95% sequenceidentity to any one of SEQ ID NOs: 3, 21, 39, 57, 77, 95, 113, 131, 149,167, 185, or 203.

It is also preferred that, the isolated antibody or antigen bindingfragment of the invention comprises a heavy chain CDR1 with the aminoacid sequence of SEQ ID NOs: 1, 19, 37, 55, 75, 93, 111, 129, 147, 165,183 or 201 or sequence variants thereof; a heavy chain CDR2 with theamino acid sequence of SEQ ID NOs: 2, 20, 38, 56, 76, 94, 112, 130, 148,166, 184, or 202 or sequence variants thereof; and a heavy chain CDR3with the amino acid sequence of SEQ ID NOs: 3, 21, 39, 57, 77, 95, 113,131, 149, 167, 185, or 203 or sequence variants thereof. In certainembodiments, an antibody or antibody fragment as provided hereincomprises a heavy chain comprising the amino acid sequence of (i) SEQ IDNO: 1 for CDRH1, SEQ ID NO: 2 for CDRH2 and SEQ ID NO: 3 for CDRH3, (ii)SEQ ID NO: 19 for CDRH1, SEQ ID NO: 20 for CDRH2, and SEQ ID NO: 21 forCDRH3, (iii) SEQ ID NO: 37 for CDRH1, SEQ ID NO: 38 for CDRH2, and SEQID NO: 39 for CDRH3, (iv) SEQ ID NO: 55 for CDRH1, SEQ ID NO: 56 forCDRH2, and SEQ ID NO: 57 for CDRH3; (v) SEQ ID NO: 75 for CDRH1, SEQ IDNO: 76 for CDRH2, and SEQ ID NO: 77 for CDRH3; (vi) SEQ ID NO: 93 forCDRH1, SEQ ID NO: 94 for CDRH2, and SEQ ID NO: 95 for CDRH3; (vii) SEQID NO: 111 for CDRH1, SEQ ID NO: 112 for CDRH2, and SEQ ID NO: 113 forCDRH3; (viii) SEQ ID NO: 129 for CDRH1, SEQ ID NO: 130 for CDRH2, andSEQ ID NO: 131 for CDRH3; (ix) SEQ ID NO: 147 for CDRH1, SEQ ID NO: 148for CDRH2, and SEQ ID NO: 149 for CDRH3; (x) SEQ ID NO: 165 for CDRH1,SEQ ID NO: 166 for CDRH2, and SEQ ID NO: 167 for CDRH3; (xi) SEQ ID NO:183 for CDRH1, SEQ ID NO: 184 for CDRH2, and SEQ ID NO: 185 for CDRH3;or (xii) SEQ ID NO: 201 for CDRH1, SEQ ID NO: 202 for CDRH2, and SEQ IDNO: 203 for CDRH3.

Preferably, the antibody or antigen binding fragment of the inventioncomprises a light chain CDR1 with the amino acid sequence of SEQ ID NOs:4, 22, 40, 58, 78, 96, 114, 132, 150, 168, 186, or 204 or sequencevariants thereof; a light chain CDR2 with the amino acid sequence of SEQID NOs: 5, 6, 23, 24, 41, 42, 59, 60, 79, 80, 97, 98, 115, 116, 133,134, 151, 152, 169, 170, 187, 188, 205, or 206 or sequence variantsthereof; and a light chain CDR3 with the amino acid sequence of SEQ IDNO: 7, 25, 43, 61, 81, 99, 117, 135, 153, 171, 189, or 201 or sequencevariants thereof. In certain embodiments, an antibody or antibodyfragment as provided herein comprises a light chain comprising the aminoacid sequence of (i) SEQ ID NO: 4 for CDRL1, SEQ ID NO: 5 or 6 forCDRL2, and SEQ ID NO: 7 for CDRL3; (ii) SEQ ID NO: 22 for CDRL1, SEQ IDNO: 23 or 24 for CDRL2, and SEQ ID NO: 25 for CDRL3; (iii) SEQ ID NO: 40for CDRL1, SEQ ID NO: 41 or 42 for CDRL2, and SEQ ID NO: 43 for CDRL3;(iv) SEQ ID NO: 58 for CDRL1, SEQ ID NO: 59 or 60 for CDRL2, and SEQ IDNO: 61 for CDRL3; (v) SEQ ID NO: 78 for CDRL1, SEQ ID NO: 79 or 80 forCDRL2, and SEQ ID NO: 81 for CDRL3; (vi) SEQ ID NO: 96 for CDRL1, SEQ IDNO: 97 or 98 for CDRL2, and SEQ ID NO: 99 for CDRL3; (vii) SEQ ID NO:114 for CDRL1, SEQ ID NO: 115 or 116 for CDRL2, and SEQ ID NO: 117 forCDRL3; (viii) SEQ ID NO: 132 for CDRL1, SEQ ID NO: 133 or 134 for CDRL2,and SEQ ID NO: 135 for CDRL3; (ix) SEQ ID NO: 150 for CDRL1, SEQ ID NO:151 or 152 for CDRL2, and SEQ ID NO: 152 for CDRL3; (x) SEQ ID NO: 168for CDRL1, SEQ ID NO: 169 or 170 for CDRL2, and SEQ ID NO: 171 forCDRL3; (xi) SEQ ID NO: 186 for CDRL1, SEQ ID NO: 187 or 188 for CDRL2,and SEQ ID NO: 189 for CDRL3; or (xii) SEQ ID NO: 204 for CDRL1, SEQ IDNO: 205 or 206 for CDRL2, and SEQ ID NO: 207 for CDRL3.

In another embodiment of the invention, the invention comprises anisolated antibody or antigen binding fragment thereof, comprising heavychain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, and CDR3 aminoacid sequences that are at least 80%, for example, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to the amino acid sequences of SEQ ID NOs:1-7, 19-25, 37-43, 55-61, 75-81, 93-99, 111-117, 129-135, 147-153,165-171, 183-189, and 201-207, respectively.

Preferably, the antibody, or the antigen binding fragment thereof, isaccording to gRVA122, gRVA144, gRVB185, gRVB492, gRVC3, gRVC20, gRVC21,gRVC38, gRVC44, gRVC58, gRVC68, or gRVC111, preferably it is accordingto gRVC20 or gRVC58. More preferably, the antibody, or the antigenbinding fragment thereof, is RVA122, RVA144, RVB185, RVB492, RVC3,RVC20, RVC21, RVC38, RVC44, RVC58, RVC68, or RVC111, preferably RVC20 orRVC58.

The present inventors have isolated twelve monoclonal antibodies (mAbs),which are referred to herein as RVA122, RVA144, RVB185, RVB492, RVC3,RVC20, RVC21, RVC38, RVC44, RVC58, RVC68, and RVC111 (cf. Examples 1 to4). Based on the antibodies RVA122, RVA144, RVB185, RVB492, RVC3, RVC20,RVC21, RVC38, RVC44, RVC58, RVC68, and RVC111, in particular on the VHand VL genes of RVA122, RVA144, RVB185, RVB492, RVC3, RVC20, RVC21,RVC38, RVC44, RVC58, RVC68, and RVC111, the terms gRVA122, gRVA144,gRVB185, gRVB492, gRVC3, gRVC20, gRVC21, gRVC38, gRVC44, gRVC58, gRVC68,or gRVC111, as used herein, refer to respective “generic” antibodies, orantigen binding fragments thereof, having the specific amino acidsequences, encoded by the specific nucleotide sequences, as outlinedbelow.

As used herein, “gRVA122” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 1, which is encoded by a CDRH1 nucleotide sequence according to SEQID NO: 8, a CDRH2 amino acid sequence according to SEQ ID NO: 2, whichis encoded by a CDRH2 nucleotide sequence according to SEQ ID NO: 9, aCDRH3 amino acid sequence according to SEQ ID NO: 3, which is encoded bya CDRH3 nucleotide sequence according to SEQ ID NO: 10, a CDRL1 aminoacid sequence according to SEQ ID NO: 4, which is encoded by a CDRL1nucleotide sequence according to SEQ ID NO: 11, a CDRL2 amino acidsequence according to SEQ ID NO: 5 or 6, which is encoded by a CDRL2nucleotide sequence according to SEQ ID NO: 12 or 13, and a CDRL3 aminoacid sequence according to SEQ ID NO: 7, which is encoded by a CDRL3nucleotide sequence according to SEQ ID NO: 14. The heavy chain variableregion (V_(H)) of “gRVA122” has an amino acid sequence according to SEQID NO: 15, which is encoded by a nucleotide sequence according to SEQ IDNO: 17, and the light chain variable region (V_(L)) of “gRVA122” has anamino acid sequence according to SEQ ID NO: 16, which is encoded by anucleotide sequence according to SEQ ID NO: 18.

As used herein, “gRVA144” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 19, which is encoded by a CDRH1 nucleotide sequence according to SEQID NO: 26, a CDRH2 amino acid sequence according to SEQ ID NO: 20, whichis encoded by a CDRH2 nucleotide sequence according to SEQ ID NO: 27, aCDRH3 amino acid sequence according to SEQ ID NO: 21, which is encodedby a CDRH3 nucleotide sequence according to SEQ ID NO: 28, a CDRL1 aminoacid sequence according to SEQ ID NO: 22, which is encoded by a CDRL1nucleotide sequence according to SEQ ID NO: 29, a CDRL2 amino acidsequence according to SEQ ID NO: 23 or 24, which is encoded by a CDRL2nucleotide sequence according to SEQ ID NO: 30 or 31, and a CDRL3 aminoacid sequence according to SEQ ID NO: 25, which is encoded by a CDRL3nucleotide sequence according to SEQ ID NO: 32. The heavy chain variableregion (V_(H)) of “gRVA144” has an amino acid sequence according to SEQID NO: 33, which is encoded by a nucleotide sequence according to SEQ IDNO: 35, and the light chain variable region (V_(L)) of “gRVA144” has anamino acid sequence according to SEQ ID NO: 34, which is encoded by anucleotide sequence according to SEQ ID NO: 36.

As used herein, “gRVB185” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 37, which is encoded by a CDRH1 nucleotide sequence according to SEQID NO: 44, a CDRH2 amino acid sequence according to SEQ ID NO: 38, whichis encoded by a CDRH2 nucleotide sequence according to SEQ ID NO: 45, aCDRH3 amino acid sequence according to SEQ ID NO: 39, which is encodedby a CDRH3 nucleotide sequence according to SEQ ID NO: 46, a CDRL1 aminoacid sequence according to SEQ ID NO: 40, which is encoded by a CDRL1nucleotide sequence according to SEQ ID NO: 47, a CDRL2 amino acidsequence according to SEQ ID NO: 41 or 42, which is encoded by a CDRL2nucleotide sequence according to SEQ ID NO: 48 or 49, and a CDRL3 aminoacid sequence according to SEQ ID NO: 43, which is encoded by a CDRL3nucleotide sequence according to SEQ ID NO: 50. The heavy chain variableregion (V_(H)) of “gRVB185” has an amino acid sequence according to SEQID NO: 51, which is encoded by a nucleotide sequence according to SEQ IDNO: 53, and the light chain variable region (V_(L)) of “gRVB185” has anamino acid sequence according to SEQ ID NO: 52, which is encoded by anucleotide sequence according to SEQ ID NO: 54.

As used herein, “gRVB492” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 55, which is encoded by a CDRH1 nucleotide sequence according to SEQID NO: 62, a CDRH2 amino acid sequence according to SEQ ID NO: 56, whichis encoded by a CDRH2 nucleotide sequence according to SEQ ID NO: 63, aCDRH3 amino acid sequence according to SEQ ID NO: 57, which is encodedby a CDRH3 nucleotide sequence according to SEQ ID NO: 64, a CDRL1 aminoacid sequence according to SEQ ID NO: 58, which is encoded by a CDRL1nucleotide sequence according to SEQ ID NO: 65, a CDRL2 amino acidsequence according to SEQ ID NO: 59 or 60, which is encoded by a CDRL2nucleotide sequence according to SEQ ID NO: 66 or 67, and a CDRL3 aminoacid sequence according to SEQ ID NO: 61, which is encoded by a CDRL3nucleotide sequence according to SEQ ID NO: 68. The heavy chain variableregion (V_(H)) of “gRVB492” has an amino acid sequence according to SEQID NO: 69 or 70, which is encoded by a nucleotide sequence according toSEQ ID NO: 72 or 73, and the light chain variable region (V_(L)) of“gRVB492” has an amino acid sequence according to SEQ ID NO: 71, whichis encoded by a nucleotide sequence according to SEQ ID NO: 74.

As used herein, “gRVC3” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 75, which is encoded by a CDRH1 nucleotide sequence according to SEQID NO: 82, a CDRH2 amino acid sequence according to SEQ ID NO: 76, whichis encoded by a CDRH2 nucleotide sequence according to SEQ ID NO: 83, aCDRH3 amino acid sequence according to SEQ ID NO: 77, which is encodedby a CDRH3 nucleotide sequence according to SEQ ID NO: 84, a CDRL1 aminoacid sequence according to SEQ ID NO: 78, which is encoded by a CDRL1nucleotide sequence according to SEQ ID NO: 85, a CDRL2 amino acidsequence according to SEQ ID NO: 79 or 80, which is encoded by a CDRL2nucleotide sequence according to SEQ ID NO: 86 or 87, and a CDRL3 aminoacid sequence according to SEQ ID NO: 81, which is encoded by a CDRL3nucleotide sequence according to SEQ ID NO: 88. The heavy chain variableregion (V_(H)) of “gRVC3” has an amino acid sequence according to SEQ IDNO: 89, which is encoded by a nucleotide sequence according to SEQ IDNO: 91, and the light chain variable region (V_(L)) of “gRVC3” has anamino acid sequence according to SEQ ID NO: 90, which is encoded by anucleotide sequence according to SEQ ID NO: 92.

As used herein, “gRVC20” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 93, which is encoded by a CDRH nucleotide sequence according to SEQID NO: 100, a CDRH2 amino acid sequence according to SEQ ID NO: 94,which is encoded by a CDRH2 nucleotide sequence according to SEQ ID NO:101, a CDRH3 amino acid sequence according to SEQ ID NO: 95, which isencoded by a CDRH3 nucleotide sequence according to SEQ ID NO: 102, aCDRL1 amino acid sequence according to SEQ ID NO: 96, which is encodedby a CDRL1 nucleotide sequence according to SEQ ID NO: 103, a CDRL2amino acid sequence according to SEQ ID NO: 97 or 98, which is encodedby a CDRL2 nucleotide sequence according to SEQ ID NO: 104 or 105, and aCDRL3 amino acid sequence according to SEQ ID NO: 99, which is encodedby a CDRL3 nucleotide sequence according to SEQ ID NO: 106. The heavychain variable region (V_(H)) of “gRVC20” has an amino acid sequenceaccording to SEQ ID NO: 107, which is encoded by a nucleotide sequenceaccording to SEQ ID NO: 109, and the light chain variable region (V_(L))of “gRVC20” has an amino acid sequence according to SEQ ID NO: 108,which is encoded by a nucleotide sequence according to SEQ ID NO: 110.

As used herein, “gRVC21” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 111, which is encoded by a CDRH1 nucleotide sequence according toSEQ ID NO: 118, a CDRH2 amino acid sequence according to SEQ ID NO: 112,which is encoded by a CDRH2 nucleotide sequence according to SEQ ID NO:119, a CDRH3 amino acid sequence according to SEQ ID NO: 113, which isencoded by a CDRH3 nucleotide sequence according to SEQ ID NO: 120, aCDRL1 amino acid sequence according to SEQ ID NO: 114, which is encodedby a CDRL1 nucleotide sequence according to SEQ ID NO: 121, a CDRL2amino acid sequence according to SEQ ID NO: 115 or 116, which is encodedby a CDRL2 nucleotide sequence according to SEQ ID NO: 122 or 123, and aCDRL3 amino acid sequence according to SEQ ID NO: 117, which is encodedby a CDRL3 nucleotide sequence according to SEQ ID NO: 124. The heavychain variable region (V_(H)) of “gRVC21” has an amino acid sequenceaccording to SEQ ID NO: 125, which is encoded by a nucleotide sequenceaccording to SEQ ID NO: 127, and the light chain variable region (V_(L))of “gRVC21” has an amino acid sequence according to SEQ ID NO: 126,which is encoded by a nucleotide sequence according to SEQ ID NO: 128.

As used herein, “gRVC38” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 129, which is encoded by a CDRH1 nucleotide sequence according toSEQ ID NO: 136, a CDRH2 amino acid sequence according to SEQ ID NO: 130,which is encoded by a CDRH2 nucleotide sequence according to SEQ ID NO:137, a CDRH3 amino acid sequence according to SEQ ID NO: 131, which isencoded by a CDRH3 nucleotide sequence according to SEQ ID NO: 138, aCDRL1 amino acid sequence according to SEQ ID NO: 132, which is encodedby a CDRL1 nucleotide sequence according to SEQ ID NO: 139, a CDRL2amino acid sequence according to SEQ ID NO: 133 or 134, which is encodedby a CDRL2 nucleotide sequence according to SEQ ID NO: 140 or 141, and aCDRL3 amino acid sequence according to SEQ ID NO: 135, which is encodedby a CDRL3 nucleotide sequence according to SEQ ID NO: 142. The heavychain variable region (V_(H)) of “gRVC38” has an amino acid sequenceaccording to SEQ ID NO: 143, which is encoded by a nucleotide sequenceaccording to SEQ ID NO: 145, and the light chain variable region (V_(L))of “gRVC38” has an amino acid sequence according to SEQ ID NO: 144,which is encoded by a nucleotide sequence according to SEQ ID NO: 146.

As used herein, “gRVC44” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 147, which is encoded by a CDRH1 nucleotide sequence according toSEQ ID NO: 154, a CDRH2 amino acid sequence according to SEQ ID NO: 148,which is encoded by a CDRH2 nucleotide sequence according to SEQ ID NO:155, a CDRH3 amino acid sequence according to SEQ ID NO: 149, which isencoded by a CDRH3 nucleotide sequence according to SEQ ID NO: 156, aCDRL1 amino acid sequence according to SEQ ID NO: 150, which is encodedby a CDRL1 nucleotide sequence according to SEQ ID NO: 157, a CDRL2amino acid sequence according to SEQ ID NO: 151 or 152, which is encodedby a CDRL2 nucleotide sequence according to SEQ ID NO: 158 or 159, and aCDRL3 amino acid sequence according to SEQ ID NO: 153, which is encodedby a CDRL3 nucleotide sequence according to SEQ ID NO: 160. The heavychain variable region (V_(H)) of “gRVC44” has an amino acid sequenceaccording to SEQ ID NO: 161, which is encoded by a nucleotide sequenceaccording to SEQ ID NO: 163, and the light chain variable region (V_(L))of “gRVC44” has an amino acid sequence according to SEQ ID NO: 162,which is encoded by a nucleotide sequence according to SEQ ID NO: 164.

As used herein, “gRVC58” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 165, which is encoded by a CDRH1 nucleotide sequence according toSEQ ID NO: 172, a CDRH2 amino acid sequence according to SEQ ID NO: 166,which is encoded by a CDRH2 nucleotide sequence according to SEQ ID NO:173, a CDRH3 amino acid sequence according to SEQ ID NO: 167, which isencoded by a CDRH3 nucleotide sequence according to SEQ ID NO: 174, aCDRL1 amino acid sequence according to SEQ ID NO: 168, which is encodedby a CDRL1 nucleotide sequence according to SEQ ID NO: 175, a CDRL2amino acid sequence according to SEQ ID NO: 169 or 170, which is encodedby a CDRL2 nucleotide sequence according to SEQ ID NO: 176 or 177, and aCDRL3 amino acid sequence according to SEQ ID NO: 171, which is encodedby a CDRL3 nucleotide sequence according to SEQ ID NO: 178. The heavychain variable region (V_(H)) of “gRVC58” has an amino acid sequenceaccording to SEQ ID NO: 179, which is encoded by a nucleotide sequenceaccording to SEQ ID NO: 181, and the light chain variable region (V_(L))of “gRVC58” has an amino acid sequence according to SEQ ID NO: 180,which is encoded by a nucleotide sequence according to SEQ ID NO: 182.

As used herein, “gRVC68” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 183, which is encoded by a CDRH1 nucleotide sequence according toSEQ ID NO: 190, a CDRH2 amino acid sequence according to SEQ ID NO: 184,which is encoded by a CDRH2 nucleotide sequence according to SEQ ID NO:191, a CDRH3 amino acid sequence according to SEQ ID NO: 185, which isencoded by a CDRH3 nucleotide sequence according to SEQ ID NO: 192, aCDRL1 amino acid sequence according to SEQ ID NO: 186, which is encodedby a CDRL1 nucleotide sequence according to SEQ ID NO: 193, a CDRL2amino acid sequence according to SEQ ID NO: 187 or 188, which is encodedby a CDRL2 nucleotide sequence according to SEQ ID NO: 194 or 195, and aCDRL3 amino acid sequence according to SEQ ID NO: 189, which is encodedby a CDRL3 nucleotide sequence according to SEQ ID NO: 196. The heavychain variable region (V_(H)) of “gRVC68” has an amino acid sequenceaccording to SEQ ID NO: 197, which is encoded by a nucleotide sequenceaccording to SEQ ID NO: 199, and the light chain variable region (V_(L))of “gRVC68” has an amino acid sequence according to SEQ ID NO: 198,which is encoded by a nucleotide sequence according to SEQ ID NO: 200.

As used herein, “gRVC111” refers to an antibody, or antigen bindingfragment thereof, having a CDRH1 amino acid sequence according to SEQ IDNO: 201, which is encoded by a CDRH1 nucleotide sequence according toSEQ ID NO: 208, a CDRH2 amino acid sequence according to SEQ ID NO: 202,which is encoded by a CDRH2 nucleotide sequence according to SEQ ID NO:209, a CDRH3 amino acid sequence according to SEQ ID NO: 203, which isencoded by a CDRH3 nucleotide sequence according to SEQ ID NO: 210, aCDRL1 amino acid sequence according to SEQ ID NO: 204, which is encodedby a CDRL1 nucleotide sequence according to SEQ ID NO: 211, a CDRL2amino acid sequence according to SEQ ID NO: 205 or 206, which is encodedby a CDRL2 nucleotide sequence according to SEQ ID NO: 212 or 213, and aCDRL3 amino acid sequence according to SEQ ID NO: 207, which is encodedby a CDRL3 nucleotide sequence according to SEQ ID NO: 214. The heavychain variable region (V_(H)) of “gRVC111” has an amino acid sequenceaccording to SEQ ID NO: 215, which is encoded by a nucleotide sequenceaccording to SEQ ID NO: 217, and the light chain variable region (V_(L))of “gRVC111” has an amino acid sequence according to SEQ ID NO: 216,which is encoded by a nucleotide sequence according to SEQ ID NO: 218.

Preferably, the antibodies according to gRVA122, gRVA144, gRVB185,gRVB492, gRVC3, gRVC20, gRVC21, gRVC38, gRVC44, gRVC58, gRVC68, orgRVC111, preferably it is according to gRVC20 and gRVC58 are of the IgG1type.

Preferably, the isolated antibody or antigen binding fragment accordingto the present invention comprises a heavy chain comprising one or more(i.e., one, two or all three) heavy chain CDRs from gRVA122, gRVA144,gRVB185, gRVB492, gRVC3, gRVC20, gRVC21, gRVC38, gRVC44, gRVC58, gRVC68,or gRVC111, preferably it is according to gRVC20 and gRVC58.

It is also preferred that the isolated antibody or antigen bindingfragment according to the present invention comprises light chain CDRsfrom gRVA122, gRVA144, gRVB185, gRVB492, gRVC3, gRVC20, gRVC21, gRVC38,gRVC44, gRVC58, gRVC68, or gRVC111, preferably it is according to gRVC20and gRVC58.

Preferably, the isolated antibody or antigen binding fragment accordingto the present invention comprises all of the CDRs of antibody RVC20 aslisted in Table 2 or all of the CDRs of antibody RVC58 as listed inTable 2. Alternatively, the isolated antibody or antigen bindingfragment according to the present invention may also preferably all ofthe CDRs of antibody RVA122 as listed in Table 2. Alternatively, theisolated antibody or antigen binding fragment according to the presentinvention may also preferably all of the CDRs of antibody RVA144 aslisted in Table 2. Alternatively, the isolated antibody or antigenbinding fragment according to the present invention may also preferablyall of the CDRs of antibody RVB185 as listed in Table 2. Alternatively,the isolated antibody or antigen binding fragment according to thepresent invention may also preferably all of the CDRs of antibody RVB492as listed in Table 2. Alternatively, the isolated antibody or antigenbinding fragment according to the present invention may also preferablyall of the CDRs of antibody RVC3 as listed in Table 2. Alternatively,the isolated antibody or antigen binding fragment according to thepresent invention may also preferably all of the CDRs of antibody RVC21as listed in Table 2. Alternatively, the isolated antibody or antigenbinding fragment according to the present invention may also preferablyall of the CDRs of antibody RVC38 as listed in Table 2. Alternatively,the isolated antibody or antigen binding fragment according to thepresent invention may also preferably all of the CDRs of antibody RVC44as listed in Table 2. Alternatively, the isolated antibody or antigenbinding fragment according to the present invention may also preferablyall of the CDRs of antibody RVC68 as listed in Table 2. Alternatively,the isolated antibody or antigen binding fragment according to thepresent invention may also preferably all of the CDRs of antibody RVC111as listed in Table 2.

The SEQ ID numbers for the amino acid sequence for the heavy chainvariable region (V_(H)) and the light chain variable region (V_(L)) ofexemplary antibodies of the invention as well as the SEQ ID numbers forthe nucleic acid sequences encoding them are listed in Table 3.

TABLE 3 SEQ ID Numbers for V_(H) and V_(L) amino acid and nucleic acidresidues for exemplary antibodies according to the present invention.V_(H) V_(L) V_(H) V_(L) amino acid amino acid nucleic acid nucleic acidRVA122 15 16 17 18 RVA144 33 34 35 36 RVB185 51 52 53 54 RVB492 69 or 7071 72 or 73 74 RVC3 89 90 91 92 RVC20 107 108 109 110 RVC21 125 126 127128 RVC38 143 144 145 146 RVC44 161 162 163 164 RVC58 179 180 181 182RVC68 197 198 199 200 RVC111 215 216 217 218

Preferably, the isolated antibody or antigen binding fragment accordingto the present invention comprises a heavy chain variable region havingan amino acid sequence that is about 70%, 75%, 80%, 85%, 88%, 90%, 92%,95%, 96%, 97%, 98%, 99% or 100% identical to the sequence recited in anyone of SEQ ID NOs: 15, 33, 51, 69, 70, 89, 107, 125, 143, 161, 179, 197,or 215. In another embodiment, the antibody or antibody fragmentcomprises a light chain variable region having an amino acid sequencethat is about 70%, 75%, 80%, 85%, 90%, 85%, 88%, 90%, 92%, 95%, 96%,97%, 98%, 99% or 100% identical to the sequence recited in SEQ ID NOs:16, 34, 52, 71, 90, 108, 126, 144, 162, 180, 198, or 216. In yet anotherpreferred embodiment, the antibody or antibody fragment comprises aheavy chain or a light chain variable region having an amino acidsequence that is about 70%, 75%, 80%, 85%, 88%, 90%, 92%, 95%, 96%, 97%,98%, 99% or 100% identical to the sequences provided in FIGS. 22 to 33 .

FIGS. 22 to 33 show the amino acid sequences for the heavy and lightchains of antibodies RVA122, RVA144, RVB185, RVB492, RVC3, RVC20, RVC21,RVC38, RVC44, RVC58, RVC68, and RVC111, respectively, as well as thenucleic acid sequences that encode them. The amino acid sequences of theCDRs and the nucleic acid sequences that encode the CDRs are in boldtext whereas the amino acid sequences of the framework region and thenucleic acid sequences that encode the framework region are in plaintext.

Preferably, the isolated antibody or antigen binding fragment accordingto the present invention comprises (i) a heavy chain variable regionhaving at least 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to the amino acid sequence of SEQ IDNO: 15 and a light chain variable region having at least 80%, forexample, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 16; or (ii) a heavychain variable region having at least 80%, for example, 85%, 88%, 90%,92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acidsequence of SEQ ID NO: 33 and a light chain variable region having atleast 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to the amino acid sequence of SEQ ID NO: 34;(iii) or a heavy chain variable region having at least 80%, for example,85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity tothe amino acid sequence of SEQ ID NO: 51 and a light chain variableregion having at least 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 52; or (iv) a heavy chain variable region having at least80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to the amino acid sequence of SEQ ID NO: 69 and alight chain variable region having at least 80%, for example, 85%, 88%,90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 71; or (v) a heavy chain variable regionhaving at least 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to the amino acid sequence of SEQ IDNO: 70 and a light chain variable region having at least 80%, forexample, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 71; or (vi) a heavychain variable region having at least 80%, for example, 85%, 88%, 90%,92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acidsequence of SEQ ID NO: 89 and a light chain variable region having atleast 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to the amino acid sequence of SEQ ID NO: 90; or(vii) a heavy chain variable region having at least 80%, for example,85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity tothe amino acid sequence of SEQ ID NO: 107 and a light chain variableregion having at least 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 108; or (viii) a heavy chain variable region having at least80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to the amino acid sequence of SEQ ID NO: 125 and alight chain variable region having at least 80%, for example, 85%, 88%,90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 126; or (ix) a heavy chain variable regionhaving at least 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to the amino acid sequence of SEQ IDNO: 143 and a light chain variable region having at least 80%, forexample, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 144; or (x) a heavychain variable region having at least 80%, for example, 85%, 88%, 90%,92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acidsequence of SEQ ID NO: 161 and a light chain variable region having atleast 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to the amino acid sequence of SEQ ID NO: 162; or(xi) a heavy chain variable region having at least 80%, for example,85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity tothe amino acid sequence of SEQ ID NO: 179 and a light chain variableregion having at least 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%,97%, 98%, 99% or 100% sequence identity to the amino acid sequence ofSEQ ID NO: 180; or (xii) a heavy chain variable region having at least80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100%sequence identity to the amino acid sequence of SEQ ID NO: 197 and alight chain variable region having at least 80%, for example, 85%, 88%,90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the aminoacid sequence of SEQ ID NO: 198; or (xiii) a heavy chain variable regionhaving at least 80%, for example, 85%, 88%, 90%, 92%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to the amino acid sequence of SEQ IDNO: 215 and a light chain variable region having at least 80%, forexample, 85%, 88%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 216.

More preferably, the antibody or the antigen binding fragment accordingto the present invention comprises: (i) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 15 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 16; or(ii) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 33 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 34; or (iii) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 51 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 52; or(iv) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 69 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 71; or (v) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 70 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 71; or(vi) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 89 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 90; or (vii) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 107 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 108; or(viii) a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 125 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 126; or (ix) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 143 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 144; or(x) a heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 161 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 162; or (xi) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 179 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 180; or(xii) a heavy chain variable region comprising the amino acid sequenceof SEQ ID NO: 197 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 198; or (xiii) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 215 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 216.

Examples of antibodies of the invention include, but are not limited to,RVA122, RVA144, RVB185, RVB492, RVC3, RVC20, RVC21, RVC38, RVC44, RVC58,RVC68, or RVC111, preferably RVC20 or RVC58. Preferably, the antibody,or the antigen binding fragment thereof, is according to gRVA122,gRVA144, gRVB185, gRVB492, gRVC3, gRVC20, gRVC21, gRVC38, gRVC44,gRVC58, gRVC68, or gRVC111, preferably it is according to gRVC20 orgRVC58.

The antibody, or the antigen binding fragment thereof, according to thepresent invention may be used in the prophylaxis, treatment orattenuation of infection by RABV and/or non-RABV lyssaviruses,preferably RABV and/or non-RABV phylogroup I lyssaviruses, morepreferably RABV and/or EBLV-i. Further details for this use aredescribed below, e.g. directly below and in the context of apharmaceutical composition and of a medical use.

Preferably, the use of the antibody, or the antigen binding fragmentthereof according to the present invention as described above, comprisesadministering said antibody in combination with another isolatedmonoclonal antibody that neutralizes lyssavirus infection and thatcomprises a heavy chain CDRH3 comprising an amino acid sequence that isat least 80%, preferably at least 90%, identical to SEQ ID NO: 95 or toSEQ ID NO: 167 and wherein both antibodies bind specifically todifferent epitopes on the glycoprotein G of RABV. The other antibody,i.e. the antibody that neutralizes lyssavirus infection and thatcomprises a heavy chain CDRH3 comprising an amino acid sequence that isat least 80%, preferably at least 90%, identical to SEQ ID NO: 95 or toSEQ ID NO: 167, is preferably also an antibody according to the presentinvention.

Preferably, the other antibody, i.e. the antibody that neutralizeslyssavirus infection and that comprises a heavy chain CDRH3 comprisingan amino acid sequence that is at least 80%, preferably at least 90%,identical to SEQ ID NO: 95 or to SEQ ID NO: 167, comprises preferably(i) heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and lightchain CDRL1, CDRL2, and CDRL3 amino acid sequences that are at least80%, preferably at least 90%, identical to the amino acid sequences ofSEQ ID NOs: 93-97 and 99 or to the amino acid sequences of SEQ ID NOs:93-96 and 98-99, respectively, or (ii) heavy chain CDRH1, CDRH2, andCDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences that are at least 80%, preferably at least 90%, identicalto the amino acid sequences of SEQ ID NOs: 165-169 and 171 or to theamino acid sequences of SEQ ID NOs: 165-168 and 170-171, respectively.More preferably, the other antibody, i.e. the antibody that neutralizeslyssavirus infection and that comprises a heavy chain CDRH3 comprisingan amino acid sequence that is at least 80%, preferably at least 90%,identical to SEQ ID NO: 95 or to SEQ ID NO: 167, comprises a heavy chainvariable region having at least 80%, preferably at least 90%, sequenceidentity to the amino acid sequence of SEQ ID NO: 107 or of SEQ ID NO:179. Even more preferably, the other antibody, i.e. the antibody thatneutralizes lyssavirus infection and that comprises a heavy chain CDRH3comprising an amino acid sequence that is at least 80%, preferably atleast 90%, identical to SEQ ID NO: 95 or to SEQ ID NO: 167, comprises(i) a heavy chain variable region having at least 80% sequence identityto the amino acid sequence of SEQ ID NO: 107 and a light chain variableregion having at least 80% sequence identity to the amino acid sequenceof SEQ ID NO: 108; or (ii) a heavy chain variable region having at least80% sequence identity to the amino acid sequence of SEQ ID NO: 179 and alight chain variable region having at least 80% sequence identity to theamino acid sequence of SEQ ID NO: 180.

Thereby, a combination of antibodies is provided for the prophylaxis,treatment or attenuation of infection by RABV and/or non-RABVlyssaviruses, preferably RABV and/or non-RABV phylogroup I lyssaviruses,more preferably RABV and/or EBLV-1, with at least one broadly antibody,including EBLV-1 neutralization.

Preferably, the antibody, or the antigen binding fragment thereof, foruse according to the present invention as described herein, binds toantigenic site I or III on the glycoprotein G of RABV and the otherantibody, which is administered in combination, binds to the other ofantigenic sites I or III on the glycoprotein G of RABV.

Interestingly, human anti-rabies antibodies most often recognizeantigenic sites I or III on the glycoprotein G of RABV, whereas forexample mouse anti-rabies antibodies most often recognize antigenic siteII on the glycoprotein G of RABV. It is assumed that the immunogenicdominance of antigenic site II is lower in humans than in mice (Kramer RA, Marissen W E, Goudsmit J, Visser T J, Clijsters-Van der Horst M,Bakker A Q, de Jong M, Jongeneelen M, Thijsse S, Backus H H, Rice A B,Weldon W C, Rupprecht C E, Dietzschold B, Bakker A B, de Kruif J (2005)The human antibody repertoire specific for rabies virus glycoprotein asselected from immune libraries. Eur J Immunol. 35(7):2131-45).Accordingly, a combination of antibodies recognizing antigenic sites Iand III on the glycoprotein G of RABV are believed to be more effectivethan for example a combination involving an antibody recognizingantigenic site II on the glycoprotein G of RABV.

Preferably, the antibody, or the antigen binding fragment thereof, foruse according to the present invention as described herein, comprises aheavy chain CDRH3 comprising an amino acid sequence that is at least80%, preferably at least 90%, identical to SEQ ID NO: 95 and wherein theother antibody administered in combination comprises a heavy chain CDRH3comprising an amino acid sequence that is at least 80%, preferably atleast 90%, identical to SEQ ID NO: 167.

Preferably, the antibody, or the antigen binding fragment thereof, foruse according to the present invention as described herein, comprisesheavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 93-97 and 99 or to the amino acid sequences of SEQ ID NOs: 93-96and 98-99, respectively, and the other antibody administered incombination comprises heavy chain CDRH1, CDRH2, and CDRH3 amino acidsequences and light chain CDRL1, CDRL2, and CDRL3 amino acid sequencesthat are at least 80%, preferably at least 90%, identical to the aminoacid sequences of SEQ ID NOs: 165-169 and 171 or to the amino acidsequences of SEQ ID NOs: 165-168 and 170-171, respectively. Morepreferably, the antibody, or the antigen binding fragment thereof, foruse according to the present invention as described herein, comprises aheavy chain variable region having at least 80% sequence identity to theamino acid sequence of SEQ ID NO: 107 and a light chain variable regionhaving at least 80% sequence identity to the amino acid sequence of SEQID NO: 108 and the other antibody administered in combination comprisesa heavy chain variable region having at least 80% sequence identity tothe amino acid sequence of SEQ ID NO: 179 and a light chain variableregion having at least 80% sequence identity to the amino acid sequenceof SEQ ID NO: 180. Even more preferably, the antibody, or the antigenbinding fragment thereof, for use according to the present invention asdescribed herein, is according to gRVC20 and wherein the other antibodyadministered in combination is according to gRVC58.

Preferably, the antibody, or the antigen binding fragment thereof, foruse according to the present invention as described herein and the otherantibody, which is administered in combination, are administeredsimultaneously or consecutively.

Of note, the antibody, or the antigen binding fragment thereof, for useaccording to the present invention as described herein and the otherantibody, which is administered in combination, may be administeredseparately, for example in separate pharmaceutical compositions, ortogether, i.e. as antibody “cocktail”, for example in the samepharmaceutical composition.

Preferably, the antibody, or the antigen binding fragment thereof, foruse according to the present invention as described herein and the otherantibody, which is administered in combination, are administeredseparately. Thereby, the antibody, or the antigen binding fragmentthereof, for use according to the present invention as described hereinand the other antibody, which is administered in combination, may beadministered either simultaneously or consecutively. For consecutiveadministration of both antibodies the time between (the end of)administration of the first antibody and (the beginning of)administration of the second antibody, e.g. in post-exposure prophylaxisor treatment of lyssavirus infection, is preferably no more than oneweek, more preferably no more than 5 days, even more preferably no morethan 3 days, most preferably no more than 2 days and particularlypreferably no more than one day (24 h). In a preferred embodimentthereof, the time between (the end of) administration of the firstantibody and (the beginning of) administration of the second antibody isno more than 12 h, preferably no more than 6 h, more preferably no morethan 3 h, even more preferably no more than 1 h, most preferably no morethan 30 min and particularly preferably no more than 10 min. Aconsecutive administration wherein one antibody is administeredimmediately after administration of the other antibody is particularlypreferred (this means in particular that the administration of one ofthe antibodies begins less than 10 min after the end of administrationof the other antibody). In consecutive administration as described aboveit is preferred that the antibody, or the antigen binding fragmentthereof, for use according to the present invention as described hereinis administered first and the other antibody, which is administered incombination, is administered thereafter. In consecutive administrationas described above it is also preferred that the other antibody, whichis administered in combination, is administered first and the antibody,or the antigen binding fragment thereof, for use according to thepresent invention as described herein is administered thereafter. In onepreferred embodiment thereof, an antibody, which is according to gRVC20,is administered first and an antibody, which is according to gRVC58, isadministered thereafter as described above. In another preferredembodiment thereof, an antibody, which is according to gRVC58, isadministered first and an antibody, which is according to gRVC20, isadministered thereafter as described above.

It is also preferred that the antibody, or the antigen binding fragmentthereof, for use according to the present invention as described hereinand the other antibody, which is administered in combination, areadministered as an antibody cocktail, e.g. in the same pharmaceuticalcomposition as described herein.

Preferably, the antibody, or the antigen binding fragment thereof, foruse according to the present invention as described herein and the otherantibody, which is administered in combination, are administered atequimolar amounts.

The invention further comprises an antibody, or fragment thereof, thatbinds to the same epitope as an antibody or antigen binding fragment ofthe invention, or an antibody that competes with an antibody or antigenbinding fragment of the invention.

Preferably, this epitope is antigenic site III.2, B, or C of the RABV Gprotein (glycoprotein G). Neither of the antibodies CR57 and CR4098binds to these epitopes, but they bind to antigenic site I and III,respectively. However, several antibodies according to the presentinvention, namely RVB181, RVC56, RVB185, RVC21, RVB161 and RVC111 bindto antigenic site III.2 of RABV G protein. The novel antigenic siteIII.2 is likely proximal to antigenic site III on the RABV G protein(cf. Example 3). Following the same criteria three additional novelantigenic sites were defined named A, B and C. Site B is defined byantibody RVC44, whose binding is not blocked by any other antibody ofthe panel. Similarly, site C is defined by antibodies RVB143 and RVC68,which also recognize a unique and distinct epitope as compared to allthe other antibodies. Of note, RVC44, RVB143 and RVC68 are the onlyantibodies of this panel capable of binding by western blot to G proteinunder reducing conditions, suggesting that they recognize a linearepitope on RABV G protein (cf. Example 3).

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention that binds to the same epitope as theantibody as described above, also neutralizes infection of RABV andinfection of at least 50% of the non-RABV lyssaviruses DUVV, EBLV-1,EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV and WCBV with anIC₅₀ below 10000 ng/ml, whereby the preferred embodiments describedabove for the antibody, or the antigen binding fragment thereof,according to the present invention that neutralizes infection of RABVand infection of at least 50% of the non-RABV lyssaviruses DUVV, EBLV-1,EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV, MOK, SHIV, BBLV and WCBV with anIC₅₀ below 10000 ng/ml also apply to the antibody, or the antigenbinding fragment thereof, according to the present invention that bindsto the same epitope as the antibody as described above.

Antibodies of the invention also include hybrid antibody molecules thatneutralize infection of RABV and infection of at least 50% of thenon-RABV lyssaviruses DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, LBV,MOK, SHIV, BBLV and WCBV with an IC₅₀ below 10000 ng/ml as describedabove and that comprise one or more CDRs from an antibody of theinvention and one or more CDRs from another antibody to the sameepitope. In one embodiment, such hybrid antibodies comprise three CDRsfrom an antibody of the invention and three CDRs from another antibodyto the same epitope. Exemplary hybrid antibodies comprise (i) the threeheavy chain CDRs from an antibody of the invention and the three lightchain CDRs from another antibody to the same epitope, or (ii) the threelight chain CDRs from an antibody of the invention and the three heavychain CDRs from another antibody to the same epitope.

In another aspect, the invention also provides a nucleic acid moleculecomprising a polynucleotide encoding the antibody, or the antigenbinding fragment thereof, according to the present invention asdescribed above. Nucleic acid sequences encoding part or all of thelight and heavy chains and CDRs of the antibodies of the presentinvention are preferred. Preferably provided herein are thus nucleicacid sequences encoding part or all of the light and heavy chains andCDRs of exemplary antibodies of the invention. The above table 3provides the SEQ ID numbers for the nucleic acid sequences encoding theheavy chain and light chain variable regions of some examples ofantibodies of the invention. Table 4 below provides the SEQ ID numbersfor the nucleic acid sequences encoding the CDRs of some examples of theantibodies of the invention. Due to the redundancy of the genetic code,variants of these nucleic acid sequences will exist that encode the sameamino acid sequences.

Thus, the present invention also comprises a nucleic acid moleculecomprising a polynucleotide encoding the antibody, or the antigenbinding fragment thereof, according to the present invention.

A nucleic acid molecule is a molecule comprising, preferably consistingof nucleic acid components. The term nucleic acid molecule preferablyrefers to DNA or RNA molecules. It is preferably used synonymous withthe term “polynucleotide”. Preferably, a nucleic acid molecule is apolymer comprising or consisting of nucleotide monomers which arecovalently linked to each other by phosphodiester-bonds of asugar/phosphate-backbone. The term “nucleic acid molecule” alsoencompasses modified nucleic acid molecules, such as base-modified,sugar-modified or backbone-modified etc. DNA or RNA molecules.

TABLE 4 SEQ ID Numbers for CDR Polynucleotides of exemplary antibodiesaccording to the present invention. SEQ ID NOs. for CDR PolynucleotidesCDRH1 CDRH2 CDRH3 CDRL1 CDRL2 CDRL3 RVA122 8 9 10 11 12 or 13 14 RVA14426 27 28 29 30 or 31 32 RVB185 44 45 46 47 48 or 49 50 RVB492 62 63 6465 66 or 67 68 RVC3 82 83 84 85 86 or 87 88 RVC20 100 101 102 103 104 or105 106 RVC21 118 119 120 121 122 or 123 124 RVC38 136 137 138 139 140or 141 142 RVC44 154 155 156 157 158 or 159 160 RVC58 172 173 174 175176 or 177 178 RVC68 190 191 192 193 194 or 195 196 RVC111 208 209 210211 212 or 213 214

Preferably, the polynucleotide sequence of the nucleic acid moleculeaccording to the invention is at least 75% identical to the nucleic acidsequence of any one of SEQ ID NOs: 8-14, 17, 18, 26-32, 35, 36, 44-50,53, 54, 62-68, 72-74, 82-88, 91, 92, 100-106, 109, 110, 118-124, 127,128, 136-142, 145, 146, 154-160, 163, 164, 172-178, 181, 182, 190-196,199, 200, 208-214, 217 or 218. Preferably, the nucleotide sequence ofthe nucleic acid molecule according to the invention is according to anyone of SEQ ID NOs: 8-14, 17, 18, 26-32, 35, 36, 44-50, 53, 54, 62-68,72-74, 82-88, 91, 92, 100-106, 109, 110, 118-124, 127, 128, 136-142,145, 146, 154-160, 163, 164, 172-178, 181, 182, 190-196, 199, 200,208-214, 217 or 218, or sequence variants thereof.

It is also preferred that, nucleic acid sequences according to theinvention include nucleic acid sequences having at least 70%, at least75%, at least 80%, at least 85%, at least 88%, at least 90%, at least92%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% identity to the nucleic acid encoding the variable region of a heavyor light chain of an antibody of the invention. In another embodiment, anucleic acid sequence of the invention has the sequence of a nucleicacid encoding a heavy or light chain CDR of an antibody of theinvention. For example, a nucleic acid sequence according to theinvention comprises a sequence that is at least 75%, at least 80%, atleast 85%, at least 88%, at least 90%, at least 92%, at least 95%, atleast 96%, at least 97%, at least 98% or at least 99% identical to thenucleic acid sequences of SEQ ID NOs: 8-14, 17, 18, 26-32, 35, 36,44-50, 53, 54, 62-68, 72-74, 82-88, 91, 92, 100-106, 109, 110, 118-124,127, 128, 136-142, 145, 146, 154-160, 163, 164, 172-178, 181, 182,190-196, 199, 200, 208-214, 217 or 218.

In yet another preferred embodiment, nucleic acid sequences according tothe invention include nucleic acid sequences having at least 70%, atleast 75%, at least 80%, at least 85%, at least 88%, at least 90%, atleast 92%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% identity to the nucleic acid encoding a heavy or light chainof an antibody of the invention as provided in FIGS. 22 to 33 .

In general, the nucleic acid molecule may be manipulated to insert,delete or alter certain nucleic acid sequences. Changes from suchmanipulation include, but are not limited to, changes to introducerestriction sites, to amend codon usage, to add or optimizetranscription and/or translation regulatory sequences, etc. It is alsopossible to change the nucleic acid to alter the encoded amino acids.For example, it may be useful to introduce one or more (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions, deletions and/orinsertions into the antibody's amino acid sequence. Such point mutationscan modify effector functions, antigen-binding affinity,post-translational modifications, immunogenicity, etc., can introduceamino acids for the attachment of covalent groups (e.g., labels) or canintroduce tags (e.g., for purification purposes). Mutations can beintroduced in specific sites or can be introduced at random, followed byselection (e.g., molecular evolution). For instance, one or more nucleicacids encoding any of the CDR regions, heavy chain variable regions orlight chain variable regions of antibodies of the invention can berandomly or directionally mutated to introduce different properties inthe encoded amino acids. Such changes can be the result of an iterativeprocess wherein initial changes are retained and new changes at othernucleotide positions are introduced. Further, changes achieved inindependent steps may be combined. Different properties introduced intothe encoded amino acids may include, but are not limited to, enhancedaffinity.

Further included within the scope of the invention are vectors, forexample, expression vectors, comprising a nucleic acid sequenceaccording to the invention. Preferably, a vector comprises a nucleicacid molecule according to the invention, for example a nucleic acidmolecule as described above.

The term “vector” refers to a nucleic acid molecule, preferably to anartificial nucleic acid molecule, i.e. a nucleic acid molecule whichdoes not occur in nature. A vector in the context of the presentinvention is suitable for incorporating or harboring a desired nucleicacid sequence. Such vectors may be storage vectors, expression vectors,cloning vectors, transfer vectors etc. A storage vector is a vectorwhich allows the convenient storage of a nucleic acid molecule. Thus,the vector may comprise a sequence corresponding, e.g., to a desiredantibody or antibody fragment thereof according to the presentinvention. An expression vector may be used for production of expressionproducts such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.For example, an expression vector may comprise sequences needed fortranscription of a sequence stretch of the vector, such as a promotersequence. A cloning vector is typically a vector that contains a cloningsite, which may be used to incorporate nucleic acid sequences into thevector. A cloning vector may be, e.g., a plasmid vector or abacteriophage vector. A transfer vector may be a vector which issuitable for transferring nucleic acid molecules into cells ororganisms, for example, viral vectors. A vector in the context of thepresent invention may be, e.g., an RNA vector or a DNA vector.Preferably, a vector is a DNA molecule. For example, a vector in thesense of the present application comprises a cloning site, a selectionmarker, such as an antibiotic resistance factor, and a sequence suitablefor multiplication of the vector, such as an origin of replication.Preferably, a vector in the context of the present application is aplasmid vector.

Cells transformed with such vectors are also included within the scopeof the invention. Examples of such cells include but are not limited to,eukaryotic cells, e.g., yeast cells, animal cells or plant cells. In oneembodiment the cells are mammalian, e.g., human, CHO, HEK293T, PER.C6,NS0, myeloma or hybridoma cells. Accordingly, the present invention alsorelates to a cell expressing the antibody, or the antigen bindingfragment thereof, according to the present invention; or comprising thevector according to the present invention.

In particular, the cell may be transfected with a vector according tothe present invention, preferably with an expression vector. The term“transfection” refers to the introduction of nucleic acid molecules,such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably intoeukaryotic cells. In the context of the present invention, the term“transfection” encompasses any method known to the skilled person forintroducing nucleic acid molecules into cells, preferably intoeukaryotic cells, such as into mammalian cells. Such methods encompass,for example, electroporation, lipofection, e.g. based on cationic lipidsand/or liposomes, calcium phosphate precipitation, nanoparticle basedtransfection, virus based transfection, or transfection based oncationic polymers, such as DEAE-dextran or polyethylenimine etc.Preferably, the introduction is non-viral.

The invention also relates to monoclonal antibodies that bind to anepitope capable of binding the antibodies or antigen binding fragmentsof the invention. Accordingly, the present invention comprises anisolated or purified immunogenic polypeptide comprising an epitope thatspecifically binds to the antibody, or the antigen binding fragmentthereof, according to the present invention. Such an immunogenic peptideaccording to the present invention may also be used for a vaccine, forexample for a vaccine which is used in combination with the antibodiesaccording to the present invention.

The invention provides novel epitopes to which the neutralizingantibodies of the invention bind. These epitopes, in particularantigenic site III.2, B and C, are found on the RABV G protein(glycoprotein G) as described above.

The epitopes to which the antibodies of the invention bind may be linear(continuous) or conformational (discontinuous). Preferably, theantibodies and antibody fragments of the invention bind a conformationalepitope, more preferably the conformational epitope is present onlyunder non-reducing conditions. However, in particular with regard toantigenic sites B and C as described above, antibodies and antibodyfragments of the invention may also bind a linear epitope, morepreferably the linear epitope is present under both, non-reducingconditions and reducing conditions.

Monoclonal and recombinant antibodies are particularly useful inidentification and purification of the individual polypeptides or otherantigens against which they are directed. The antibodies of theinvention have additional utility in that they may be employed asreagents in immunoassays, radioimmunoassays (RIA) or enzyme-linkedimmunosorbent assays (ELISA). In these applications, the antibodies canbe labeled with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme. The antibodies mayalso be used for the molecular identification and characterization(epitope mapping) of antigens.

Antibodies of the invention can also be coupled to a drug for deliveryto a treatment site or coupled to a detectable label to facilitateimaging of a site comprising cells of interest, such as cells infectedwith RABV and/or non-RABV lyssaviruses. Methods for coupling antibodiesto drugs and detectable labels are well known in the art, as are methodsfor imaging using detectable labels. Labeled antibodies may be employedin a wide variety of assays, employing a wide variety of labels.Detection of the formation of an antibody-antigen complex between anantibody of the invention and an epitope of interest (an epitope of RABVand/or non-RABV) can be facilitated by attaching a detectable substanceto the antibody. Suitable detection means include the use of labels suchas radionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors,prosthetic group complexes, free radicals, particles, dyes, and thelike. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material is luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin; and examples ofsuitable radioactive material include 125I, 131I, 35S, or 3H. Suchlabeled reagents may be used in a variety of well-known assays, such asradioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescentimmunoassays, and the like. (See U.S. Pat. Nos. 3,766,162; 3,791,932;3,817,837; and 4,233,402 for example).

An antibody according to the invention may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent, or aradioactive metal ion or radioisotope. Examples of radioisotopesinclude, but are not limited to, 1-131, 1-123, 1-125, Y-90, Re-188,Re-186, At-211, Cu-67, Bi-212, Bi-213, Pd-109, Tc-99, In-111, and thelike. Such antibody conjugates can be used for modifying a givenbiological response; the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known. See, for example, Arnon et al. (1985) “Monoclonal Antibodiesfor Immunotargeting of Drugs in Cancer Therapy,” in MonoclonalAntibodies and Cancer Therapy, ed. Reisfeld et al. (Alan R. Liss, Inc.),pp. 243-256; ed. Hellstrom et al. (1987) “Antibodies for Drug Delivery,”in Controlled Drug Delivery, ed. Robinson et al. (2d ed; Marcel Dekker,Inc.), pp. 623-653; Thorpe (1985) “Antibody Carriers of Cytotoxic Agentsin Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biologicaland Clinical Applications, ed. Pinchera et al. pp. 475-506 (EditriceKurtis, Milano, Italy, 1985); “Analysis, Results, and Future Prospectiveof the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy,” inMonoclonal Antibodies for Cancer Detection and Therapy, ed. Baldwin etal. (Academic Press, New York, 1985), pp. 303-316; and Thorpe et al.(1982) Immunol. Rev. 62:119-158.

Alternatively, an antibody, or antibody fragment thereof, can beconjugated to a second antibody, or antibody fragment thereof, to forman antibody heteroconjugate as described in U.S. Pat. No. 4,676,980. Inaddition, linkers may be used between the labels and the antibodies ofthe invention (e.g., U.S. Pat. No. 4,831,175). Antibodies or,antigen-binding fragments thereof may be directly labeled withradioactive iodine, indium, yttrium, or other radioactive particle knownin the art (e.g., U.S. Pat. No. 5,595,721). Treatment may consist of acombination of treatment with conjugated and non-conjugated antibodiesadministered simultaneously or subsequently (e.g., WO00/52031;WO00/52473).

Antibodies of the invention may also be attached to a solid support.Additionally, antibodies of the invention, or functional antibodyfragments thereof, can be chemically modified by covalent conjugation toa polymer to, for example, increase their circulating half-life.Examples of polymers, and methods to attach them to peptides, are shownin U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546. In someembodiments the polymers may be selected from polyoxyethylated polyolsand polyethylene glycol (PEG). PEG is soluble in water at roomtemperature and has the general formula: R(O—CH2-CH2)_(n)O—R where R canbe hydrogen, or a protective group such as an alkyl or alkanol group.Preferably, the protective group may have between 1 and 8 carbons. Forexample, the protective group is methyl. The symbol n is a positiveinteger. In one embodiment n is between 1 and 1,000. In anotherembodiment n is between 2 and 500. Preferably, the PEG has an averagemolecular weight between 1,000 and 40,000, more preferably the PEG has amolecular weight between 2,000 and 20,000, even more preferably the PEGhas a molecular weight between 3,000 and 12,000. Furthermore, PEG mayhave at least one hydroxy group, for example the PEG may have a terminalhydroxy group. For example, it is the terminal hydroxy group which isactivated to react with a free amino group on the inhibitor. However, itwill be understood that the type and amount of the reactive groups maybe varied to achieve a covalently conjugated PEG/antibody of the presentinvention.

Water-soluble polyoxyethylated polyols are also useful in the presentinvention. They include polyoxyethylated sorbitol, polyoxyethylatedglucose, polyoxyethylated glycerol (POG), and the like. In oneembodiment, POG is used. Without being bound by any theory, because theglycerol backbone of polyoxyethylated glycerol is the same backboneoccurring naturally in, for example, animals and humans in mono-, di-,triglycerides, this branching would not necessarily be seen as a foreignagent in the body. POG may have a molecular weight in the same range asPEG. Another drug delivery system that can be used for increasingcirculatory half-life is the liposome. Methods of preparing liposomedelivery systems are known to one of skill in the art. Other drugdelivery systems are known in the art and are described in, for example,referenced in Poznansky et al. (1980) and Poznansky (1984).

Antibodies of the invention may be provided in purified form. Typically,the antibody will be present in a composition that is substantially freeof other polypeptides e.g., where less than 90% (by weight), usuallyless than 60% and more usually less than 50% of the composition is madeup of other polypeptides.

Antibodies of the invention may be immunogenic in non-human (orheterologous) hosts e.g., in mice. In particular, the antibodies mayhave an idiotope that is immunogenic in non-human hosts, but not in ahuman host. Antibodies of the invention for human use include those thatcannot be easily isolated from hosts such as mice, goats, rabbits, rats,non-primate mammals, etc. and cannot generally be obtained byhumanization or from xeno-mice.

Antibodies of the invention can be of any isotype (e.g., IgA, IgG, IgMi.e. an α, γ or μ heavy chain), but will preferably be IgG. Within theIgG isotype, antibodies may be IgG1, IgG2, IgG3 or IgG4 subclass,whereby IgG1 is preferred. Antibodies of the invention may have a κ or aλ light chain.

Production of Antibodies

Antibodies according to the invention can be made by any method known inthe art. For example, the general methodology for making monoclonalantibodies using hybridoma technology is well known (Kohler, G. andMilstein, C. 1975; Kozbar et al. 1983). In one embodiment, thealternative EBV immortalization method described in WO2004/076677 isused.

Using the method described in WO 2004/076677, B cells producing theantibody of the invention can be transformed with EBV and a polyclonal Bcell activator. Additional stimulants of cellular growth anddifferentiation may optionally be added during the transformation stepto further enhance the efficiency. These stimulants may be cytokinessuch as IL-2 and IL-15. In one aspect, IL-2 is added during theimmortalization step to further improve the efficiency ofimmortalization, but its use is not essential. The immortalized B cellsproduced using these methods can then be cultured using methods known inthe art and antibodies isolated therefrom.

Using the method described in WO 2010/046775, plasma cells can becultured in limited numbers, or as single plasma cells in microwellculture plates. Antibodies can be isolated from the plasma cellcultures. Further, from the plasma cell cultures, RNA can be extractedand PCR can be performed using methods known in the art. The VH and VLregions of the antibodies can be amplified by RT-PCR (reversetranscriptase PCR), sequenced and cloned into an expression vector thatis then transfected into HEK293T cells or other host cells. The cloningof nucleic acid in expression vectors, the transfection of host cells,the culture of the transfected host cells and the isolation of theproduced antibody can be done using any methods known to one of skill inthe art.

The antibodies may be further purified, if desired, using filtration,centrifugation and various chromatographic methods such as HPLC oraffinity chromatography. Techniques for purification of antibodies,e.g., monoclonal antibodies, including techniques for producingpharmaceutical-grade antibodies, are well known in the art.

Fragments of the antibodies of the invention can be obtained from theantibodies by methods that include digestion with enzymes, such aspepsin or papain, and/or by cleavage of disulfide bonds by chemicalreduction. Alternatively, fragments of the antibodies can be obtained bycloning and expression of part of the sequences of the heavy or lightchains. Antibody “fragments” include Fab, Fab′, F(ab′)2 and Fvfragments. The invention also encompasses single-chain Fv fragments(scFv) derived from the heavy and light chains of an antibody of theinvention. For example, the invention includes a scFv comprising theCDRs from an antibody of the invention. Also included are heavy or lightchain monomers and dimers, single domain heavy chain antibodies, singledomain light chain antibodies, as well as single chain antibodies, e.g.,single chain Fv in which the heavy and light chain variable domains arejoined by a peptide linker.

Antibody fragments of the invention may impart monovalent or multivalentinteractions and be contained in a variety of structures as describedabove. For instance, scFv molecules may be synthesized to create atrivalent “triabody” or a tetravalent “tetrabody.” The scFv moleculesmay include a domain of the Fc region resulting in bivalent minibodies.In addition, the sequences of the invention may be a component ofmultispecific molecules in which the sequences of the invention targetthe epitopes of the invention and other regions of the molecule bind toother targets. Exemplary molecules include, but are not limited to,bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies(Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

Standard techniques of molecular biology may be used to prepare DNAsequences encoding the antibodies or antibody fragments of the presentinvention. Desired DNA sequences may be synthesized completely or inpart using oligonucleotide synthesis techniques. Site-directedmutagenesis and polymerase chain reaction (PCR) techniques may be usedas appropriate.

Any suitable host cell/vector system may be used for expression of theDNA sequences encoding the antibody molecules of the present inventionor fragments thereof. Bacterial, for example E. coli, and othermicrobial systems may be used, in part, for expression of antibodyfragments such as Fab and F(ab′)2 fragments, and especially Fv fragmentsand single chain antibody fragments, for example, single chain Fvs.Eukaryotic, e.g., mammalian, host cell expression systems may be usedfor production of larger antibody molecules, including complete antibodymolecules. Suitable mammalian host cells include, but are not limitedto, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell comprising a vector encoding a nucleic acid of thepresent invention under conditions suitable for expression of proteinfrom DNA encoding the antibody molecule of the present invention, andisolating the antibody molecule.

The antibody molecule may comprise only a heavy or light chainpolypeptide, in which case only a heavy chain or light chain polypeptidecoding sequence needs to be used to transfect the host cells. Forproduction of products comprising both heavy and light chains, the cellline may be transfected with two vectors, a first vector encoding alight chain polypeptide and a second vector encoding a heavy chainpolypeptide. Alternatively, a single vector may be used, the vectorincluding sequences encoding light chain and heavy chain polypeptides.

Alternatively, antibodies according to the invention may be produced by(i) expressing a nucleic acid sequence according to the invention in ahost cell, and (ii) isolating the expressed antibody product.Additionally, the method may include (iii) purifying the isolatedantibody. Transformed B cells and cultured plasma cells may be screenedfor those producing antibodies of the desired specificity or function.

The screening step may be carried out by any immunoassay, e.g., ELISA,by staining of tissues or cells (including transfected cells), byneutralization assay or by one of a number of other methods known in theart for identifying desired specificity or function. The assay mayselect on the basis of simple recognition of one or more antigens, ormay select on the additional basis of a desired function e.g., to selectneutralizing antibodies rather than just antigen-binding antibodies, toselect antibodies that can change characteristics of targeted cells,such as their signaling cascades, their shape, their growth rate, theircapability of influencing other cells, their response to the influenceby other cells or by other reagents or by a change in conditions, theirdifferentiation status, etc.

Individual transformed B cell clones may then be produced from thepositive transformed B cell culture. The cloning step for separatingindividual clones from the mixture of positive cells may be carried outusing limiting dilution, micromanipulation, single cell deposition bycell sorting or another method known in the art.

Nucleic acid from the cultured plasma cells can be isolated, cloned andexpressed in HEK293T cells or other known host cells using methods knownin the art.

The immortalized B cell clones or the transfected host-cells of theinvention can be used in various ways e.g., as a source of monoclonalantibodies, as a source of nucleic acid (DNA or mRNA) encoding amonoclonal antibody of interest, for research, etc.

The invention also provides a composition comprising immortalized Bmemory cells or transfected host cells that produce antibodies accordingto the present invention.

The immortalized B cell clone or the cultured plasma cells of theinvention may also be used as a source of nucleic acid for the cloningof antibody genes for subsequent recombinant expression. Expression fromrecombinant sources is more common for pharmaceutical purposes thanexpression from B cells or hybridomas e.g., for reasons of stability,reproducibility, culture ease, etc.

Thus the invention also provides a method for preparing a recombinantcell, comprising the steps of: (i) obtaining one or more nucleic acids(e.g., heavy and/or light chain mRNAs) from the B cell clone or thecultured plasma cells that encodes the antibody of interest; (ii)inserting the nucleic acid into an expression vector and (iii)transfecting the vector into a host cell in order to permit expressionof the antibody of interest in that host cell.

Similarly, the invention provides a method for preparing a recombinantcell, comprising the steps of: (i) sequencing nucleic acid(s) from the Bcell clone or the cultured plasma cells that encodes the antibody ofinterest; and (ii) using the sequence information from step (i) toprepare nucleic acid(s) for insertion into a host cell in order topermit expression of the antibody of interest in that host cell. Thenucleic acid may, but need not, be manipulated between steps (i) and(ii) to introduce restriction sites, to change codon usage, and/or tooptimize transcription and/or translation regulatory sequences.

Furthermore, the invention also provides a method of preparing atransfected host cell, comprising the step of transfecting a host cellwith one or more nucleic acids that encode an antibody of interest,wherein the nucleic acids are nucleic acids that were derived from animmortalized B cell clone or a cultured plasma cell of the invention.Thus the procedures for first preparing the nucleic acid(s) and thenusing it to transfect a host cell can be performed at different times bydifferent people in different places (e.g., indifferent countries).

These recombinant cells of the invention can then be used for expressionand culture purposes. They are particularly useful for expression ofantibodies for large-scale pharmaceutical production. They can also beused as the active ingredient of a pharmaceutical composition. Anysuitable culture technique can be used, including but not limited tostatic culture, roller bottle culture, ascites fluid, hollow-fiber typebioreactor cartridge, modular minifermenter, stirred tank, microcarrierculture, ceramic core perfusion, etc.

Methods for obtaining and sequencing immunoglobulingenes from B cells orplasma cells are well known in the art (e.g., see Chapter 4 of KubyImmunology, 4th edition, 2000).

The transfected host cell may be a eukaryotic cell, including yeast andanimal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells,human cells such as PER.C6 or HKB-11 cells, myeloma cells), as well asplant cells. Preferred expression hosts can glycosylate the antibody ofthe invention, particularly with carbohydrate structures that are notthemselves immunogenic in humans. In one embodiment the transfected hostcell may be able to grow inserum-free media. In a further embodiment thetransfected host cell may be able to grow in culture without thepresence of animal-derived products. The transfected host cell may alsobe cultured to give a cell line.

The invention also provides a method for preparing one or more nucleicacid molecules (e.g., heavy and light chain genes) that encode anantibody of interest, comprising the steps of: (i) preparing animmortalized B cell clone or culturing plasma cells according to theinvention; (ii) obtaining from the B cell clone or the cultured plasmacells nucleic acid that encodes the antibody of interest. Further, theinvention provides a method for obtaining a nucleic acid sequence thatencodes an antibody of interest, comprising the steps of: (i) preparingan immortalized B cell clone or culturing plasma cells according to theinvention; (ii) sequencing nucleic acid from the B cell clone or thecultured plasma cells that encodes the antibody of interest.

The invention further provides a method of preparing nucleic acidmolecule(s) that encode an antibody of interest, comprising the step ofobtaining the nucleic acid that was obtained from a transformed B cellclone or cultured plasma cells of the invention. Thus the procedures forfirst obtaining the B cell clone or the cultured plasma cell, and thenobtaining nucleic acid(s) from the B cell clone or the cultured plasmacells can be performed at different times by different people indifferent places (e.g., in different countries).

The invention also comprises a method for preparing an antibody (e.g.,for pharmaceutical use) according to the present invention, comprisingthe steps of: (i) obtaining and/or sequencing one or more nucleic acids(e.g., heavy and light chain genes) from the selected B cell clone orthe cultured plasma cells expressing the antibody of interest; (ii)inserting the nucleic acid(s) into or using the nucleic acid(s)sequence(s) to prepare an expression vector; (iii) transfecting a hostcell that can express the antibody of interest; (iv) culturing orsub-culturing the transfected host cells under conditions where theantibody of interest is expressed; and, optionally, (v) purifying theantibody of interest.

The invention also provides a method of preparing an antibody comprisingthe steps of culturing or sub-culturing a transfected host cellpopulation under conditions where the antibody of interest is expressedand, optionally, purifying the antibody of interest, wherein saidtransfected host cell population has been prepared by (i) providingnucleic acid(s) encoding a selected antibody of interest that isproduced by a B cell clone or cultured plasma cells prepared asdescribed above, (ii) inserting the nucleic acid(s) into an expressionvector, (iii) transfecting the vector in a host cell that can expressthe antibody of interest, and (iv) culturing or sub-culturing thetransfected host cell comprising the inserted nucleic acids to producethe antibody of interest. Thus the procedures for first preparing therecombinant host cell and then culturing it to express antibody can beperformed at very different times by different people in differentplaces (e.g., in different countries).

Epitopes

As mentioned above, the antibodies of the invention can be used to mapthe epitopes to which they bind. The invention provides antibodies,which bind to known epitopes and antibodies, which bind to novelepitopes on the RABV G protein (glycoprotein G). These epitopes of theRABV G Protein and examples of antibodies, which bind to each of theepitopes, are described in detail in Examples 3 and 5, and morespecifically concerning the conservation of antigenic sites I and III onthe RABV G protein in Example 6.

In general, the epitopes to which the antibodies of the invention bindmay be linear (continuous) or conformational (discontinuous). In onepreferred embodiment, the antibodies and antibody fragments of theinvention bind a conformational epitope. It is also preferred, that theconformational epitope is present only under non-reducing conditions.

Namely, the two reference antibodies CR57 and CR4098 were previouslyshown to recognize RABV G protein antigenic sites I and III (Bakker, A.B. H. et al., J Virol 79, 9062-9068, 2005), respectively. The exemplaryantibodies according to the present invention can be clustered into 6groups. RVA125, RVC3, RVC20 and RVD74 bind to the antigenic site Igroup. Of note, the binding of antigenic site I antibodies to G proteinis enhanced by a subgroup of non-antigenic site-I antibodies. RVA122,RVA144, RVB492, RVC4, RVC69, RVC38 and RVC58 bind to the antigenic siteIII, whereby the RVC58 epitope might only partially overlap withantigenic site III. A third cluster composed by antibodies RVB181,RVC56, RVB185, RVC21, RVB161 and RVC111 binds to antigenic site III.2,which is likely proximal to antigenic site III on the G protein. Threeadditional sites were defined named A, B and C. The site A is defined bythe unique antibody RVB686, whereby RVB686 binding might induce anallosteric effect on the G protein that compromises the binding of mostother antibodies. Site B is defined by antibody RVC44. Similarly, site Cis defined by antibodies RVB143 and RVC68, which also recognize a uniqueand distinct epitope as compared to all the other antibodies. Of note,RVC44, RVB143 and RVC68 are the only antibodies of this panel capable ofbinding to G protein under reducing conditions, suggesting that theyrecognize a linear epitope on RABV G protein.

The polypeptides that bind to the antibodies of the present inventionmay have a number of uses. The polypeptides and polypeptide variantsthereof in purified or synthetic form can be used to raise immuneresponses (i.e., as a vaccine, or for the production of antibodies forother uses) or for screening sera for antibodies that immunoreact withthe epitope or mimotopes thereof. In one embodiment such polypeptides orpolypeptide variants, or antigen comprising such polypeptides orpolypeptide variants may be used as a vaccine for raising an immuneresponse that comprises antibodies of the same quality as thosedescribed in the present invention.

Furthermore, the present invention also relates to the use of theantibody, or the antigen binding fragment thereof, according to thepresent invention, for monitoring the quality of anti-RABV and/oranti-non-RABV lyssavirus vaccines by checking that the antigen of saidvaccine contains the specific epitope in the correct conformation.

The antibodies and antibody fragments of the invention can also be usedin a method of monitoring the quality of vaccines. In particular theantibodies can be used to check that the antigen in a vaccine containsthe correct immunogenic epitope in the correct conformation. The use ofan antibody of the invention, or an antigen binding fragment thereof,for monitoring the quality of a vaccine against RABV and/or non-RABVlyssavirus by, for example, checking that the antigen of said vaccinecontains the specific epitope in the correct conformation is alsocontemplated to be within the scope of the invention.

The polypeptides that bind to the antibodies of the present inventionmay also be useful in screening for ligands that bind to saidpolypeptides. Such ligands, include but are not limited to antibodies;including those from camels, sharks and other species, fragments ofantibodies, peptides, phage display technology products, aptamers,adnectins or fragments of other viral or cellular proteins, may blockthe epitope and so prevent infection. Such ligands are encompassedwithin the scope of the invention.

Pharmaceutical Compositions The invention provides a pharmaceuticalcomposition comprising one or more of: the antibodies or antibodyfragments of the invention; the nucleic acid encoding such antibodies orfragments; the vector encoding the nucleic acids; the cell expressingthe antibody or comprising the vector; or the immunogenic polypeptiderecognized by the antibodies or antigen binding fragment of theinvention. The pharmaceutical composition may also contain apharmaceutically acceptable carrier or excipient. Preferably, thepharmaceutical composition comprises the antibody, or the antigenbinding fragment thereof, according to the present invention, thenucleic acid according to the present invention, the vector according tothe present invention, the cell according to the present invention, orthe immunogenic polypeptide according to the present invention, and apharmaceutically acceptable excipient, diluent or carrier.

Although the carrier or excipient may facilitate administration, itshould not itself induce the production of antibodies harmful to theindividual receiving the composition. Nor should it be toxic. Suitablecarriers may be large, slowly metabolized macromolecules such asproteins, polypeptides, liposomes, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers andinactive virus particles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the subject.

Compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g., a lyophilized composition, like Synagis™ and Herceptin™,for reconstitution with sterile water containing a preservative). Thecomposition may be prepared for topical administration e.g., as anointment, cream or powder. The composition may be prepared for oraladministration e.g., as a tablet or capsule, as a spray, or as a syrup(optionally flavored). The composition may be prepared for pulmonaryadministration e.g., as an inhaler, using a fine powder or a spray. Thecomposition may be prepared as a suppository or pessary. The compositionmay be prepared for nasal, aural or ocular administration e.g., asdrops. The composition may be in kit form, designed such that a combinedcomposition is reconstituted just prior to administration to a subject.For example, a lyophilized antibody can be provided in kit form withsterile water or a sterile buffer.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule, an antibody fragment or variants andderivatives thereof. As such, it will be susceptible to degradation inthe gastrointestinal tract. Thus, if the composition is to beadministered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Gennaro (2000) Remington: The Science and Practice ofPharmacy, 20th edition, ISBN: 0683306472.

Pharmaceutical compositions of the invention generally have a pH between5.5 and 8.5, in some embodiments this may be between 6 and 8, and inother embodiments about 7. The pH may be maintained by the use of abuffer. The composition may be sterile and/or pyrogen free. Thecomposition may be isotonic with respect to humans. In one embodimentpharmaceutical compositions of the invention are supplied inhermetically-sealed containers.

Within the scope of the invention are compositions present in severalforms of administration; the forms include, but are not limited to,those forms suitable for parenteral administration, e.g., by injectionor infusion, for example by bolus injection or continuous infusion.Where the product is for injection or infusion, it may take the form ofa suspension, solution or emulsion in an oily or aqueous vehicle and itmay contain formulatory agents, such as suspending, preservative,stabilizing and/or dispersing agents. Alternatively, the antibodymolecule may be in dry form, for reconstitution before use with anappropriate sterile liquid. A vehicle is typically understood to be amaterial that is suitable for storing, transporting, and/oradministering a compound, such as a pharmaceutically active compound, inparticular the antibodies according to the present invention. Forexample, the vehicle may be a physiologically acceptable liquid, whichis suitable for storing, transporting, and/or administering apharmaceutically active compound, in particular the antibodies accordingto the present invention. Once formulated, the compositions of theinvention can be administered directly to the subject. In one embodimentthe compositions are adapted for administration to mammalian, e.g.,human subjects.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intranasal, enteral, sublingual, intravaginal or rectalroutes. Hyposprays may also be used to administer the pharmaceuticalcompositions of the invention. Typically, the therapeutic compositionsmay be prepared as injectables, either as liquid solutions orsuspensions. Solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue,whereby intravenously or intramuscularly injection are preferred andintramuscularly injection is more preferred. The compositions can alsobe administered into a lesion. Particularly preferably, the compositionaccording to the present invention is administered similarly to knownrabies immunoglobulins (RIGs) in post-exposure prophylaxis. For example,the WHO recommends to administer all of the RIG, or as much asanatomically possible to avoid possible compartment syndrome, into oraround the wound site or sites (e.g., the bite site). The remainingimmunoglobulin, if any, should be injected intramuscularly at a sitedistant from the site of vaccine administration (cf.who.int/rabies/human/WHO_strategy_prepost_exposure/en/indexi.html#,retrieved at Nov. 12, 2014). Accordingly, this administration method isalso particularly preferred for the antibody and the pharmaceuticalcomposition according to the present invention, at least inpost-exposure prophylaxis and possibly also in treatment or otherapplications.

Dosage treatment may be a single dose schedule or a multiple doseschedule. Known antibody-based pharmaceuticals provide guidance relatingto frequency of administration e.g., whether a pharmaceutical should bedelivered daily, weekly, monthly, etc. Frequency and dosage may alsodepend on the severity of symptoms. In particular, the treatmentschedule for rabies immunoglobulins provide guidance relating tofrequency of administration. For RIGs it is recommended in post-exposureprophylaxis to administer the RIG for passive immunization only once,preferably at, or as soon as possible after, the initiation ofpost-exposure vaccination. Accordingly, this treatment schedule is alsoparticularly preferred for the antibody and the pharmaceuticalcomposition according to the present invention, at least inpost-exposure prophylaxis. In more general, i.e. for all applications,it is preferred that the antibody or the pharmaceutical compositionaccording to the present invention is administered in a single doseschedule, i.e. only one single dose. Accordingly, the pharmaceuticalcomposition is preferably provided as single-dose product, i.e. as aproduct which comprises only one single dose. However, since the dosemay depend on the bodyweight, in such cases a dose corresponding to amaximal bodyweight is considered as “single dose”.

In particular, it is preferred that the amount of the antibody, or theantigen binding fragment thereof, in the pharmaceutical compositionaccording to the present invention, does not exceed 100 mg, preferablydoes not exceed 50 mg, more preferably does not exceed 20 mg, even morepreferably does not exceed 10 mg, and particularly preferably does notexceed 5 mg. This amount of antibody preferably refers to a single doseas described above. The dose of the antibody according the presentinvention, which is effective e.g. in post-exposure prophylaxis, is thusvery low, whereas the recommended amount of HRIG is 20 IU/kg bodyweight,for ERIG and F(ab′)2 it is even 40 IU/kg bodyweight. Such a low amountof the antibody according to the present invention could be produced andformulated in a stable form (i.e. lyophilized formulation, where forinstance previous studies have shown that monoclonal antibodiespreserved by lyophilization are stable for 33 months at 40° C. and 5months at 50° C.) and at an affordable cost also for developingcountries.

However, the antibody or the pharmaceutical composition according to thepresent invention may also be administered in more than one dose, e.g.in severe cases, for example in treatment protocols.

Preferably, the composition according to the invention is administeredto a subject after an infection with a RABV and/or a non-RABV lyssavirushas taken place.

Pharmaceutical compositions will include an effective amount of one ormore antibodies of the invention and/or a polypeptide comprising anepitope that binds an antibody of the invention i.e., an amount that issufficient to treat, ameliorate, attenuate or prevent a desired diseaseor condition, or to exhibit a detectable therapeutic effect. Therapeuticeffects also include reduction or attenuation in pathogenic potency orphysical symptoms. The precise effective amount for any particularsubject will depend upon their size, weight, and health, the nature andextent of the condition, and the therapeutics or combination oftherapeutics selected for administration. The effective amount for agiven situation is determined by routine experimentation and is withinthe judgment of a clinician. For purposes of the present invention, aneffective dose will generally be from about 0.005 to about 100 mg/kg,preferably from about 0.0075 to about 75 mg/kg, more preferably fromabout 0.01 to about 60 mg/kg, even more preferably from about 0.03 toabout 50 mg/kg of the antibody of the present invention in theindividual to which it is administered.

Preferably, the pharmaceutical composition according to the presentinvention comprises at least two antibodies or antigen binding fragmentsthereof, according to the present invention, wherein the two antibodies,or the antigen binding fragments thereof, specifically bind to differentepitopes on the glycoprotein G of RABV. For example, the pharmaceuticalcomposition according to the present invention comprises a firstantibody or an antigen binding fragment thereof, according to thepresent invention, and a second antibody, or an antigen binding fragmentthereof, according to the present invention, wherein the first antibody,or the antigen binding fragment thereof, specifically binds to anotherepitope on the glycoprotein G of RABV than the second antibody or thesecond antigen binding fragment thereof.

I.e., the two antibodies according to the present invention bindspecifically to the RABV G protein, but to different epitopes on theRABV G protein. For example, one antibody may specifically bind toantigenic site I on the RABV G protein and a further antibody mayspecifically bind to an epitope on the glycoprotein G of RABV, which atleast partially overlaps with antigenic site III on the glycoprotein Gof RABV. The antigenic sites of the exemplary antibodies according tothe present invention are outlined in Example 3. Moreover, whether twoantibodies bind to the same or different epitopes on the RABV G proteinmay be easily determined by the person skilled in the art, for exampleby use of any competition study, whereby an example of a competitionstudy is shown in Example 3.

Preferably, one antibody of the at least two antibodies comprised bysuch a pharmaceutical composition according to the present inventionbinds (specifically) to antigenic site I on the glycoprotein G of RABVand another antibody of the at least two antibodies comprised by such apharmaceutical composition according to the present invention binds(specifically) to antigenic site III on the glycoprotein G of RABV.

More preferably, at least one of the at least two antibodies or antigenbinding fragments thereof comprised by such a pharmaceutical compositionaccording to the present invention comprises a heavy chain CDRH3comprising an amino acid sequence that is at least 80%, preferably atleast 90%, identical to SEQ ID NO: 95 or to SEQ ID NO: 167.

In a preferred a pharmaceutical composition according to the presentinvention one of the at least two antibodies or antigen bindingfragments thereof comprises (i) heavy chain CDRH1, CDRH2, and CDRH3amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acidsequences that are at least 80%, preferably at least 90%, identical tothe amino acid sequences of SEQ ID NOs: 93-97 and 99 or to the aminoacid sequences of SEQ ID NOs: 93-96 and 98-99, respectively or (ii)heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 165-169 and 171 or to the amino acid sequences of SEQ ID NOs:165-168 and 170-171, respectively. More preferably, one of the at leasttwo antibodies or antigen binding fragments thereof comprises a heavychain variable region having at least 80%, preferably at least 90%,sequence identity to the amino acid sequence of SEQ ID NO: 107 or of SEQID NO: 179.

In a particularly preferred pharmaceutical composition according to thepresent invention comprising at least two antibodies according to thepresent invention as described herein one of the at least two antibodiesor antigen binding fragments thereof comprises a heavy chain CDRH3comprising an amino acid sequence that is at least 80%, preferably atleast 90%, identical to SEQ ID NO: 95 and wherein another of the atleast two antibodies comprises a heavy chain CDRH3 comprising an aminoacid sequence that is at least 80%, preferably at least 90%, identicalto SEQ ID NO: 167. Preferably, one of the at least two antibodies orantigen binding fragments thereof comprises heavy chain CDRH1, CDRH2,and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3amino acid sequences that are at least 80%, preferably at least 90%,identical to the amino acid sequences of SEQ ID NOs: 93-97 and 99 or tothe amino acid sequences of SEQ ID NOs: 93-96 and 98-99, respectively,and another of the at least two antibodies comprises heavy chain CDRH1,CDRH2, and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, andCDRL3 amino acid sequences that are at least 80%, preferably at least90%, identical to the amino acid sequences of SEQ ID NOs: 165-169 and171 or to the amino acid sequences of SEQ ID NOs: 165-168 and 170-171,respectively. More preferably, one of the at least two antibodies orantigen binding fragments thereof is according to gRVC20 and whereinanother of the at least two antibodies is according to gRVC58.

A particularly preferred example of two antibodies according to thepresent invention, which specifically bind to different epitopes on theglycoprotein G of RABV, is RVC20 and RVC58.

Moreover, the pharmaceutical composition may also contain more than two,e.g. 3, 4, 5, 6, etc., antibodies according to the present invention,whereby at least two, preferably more than two, more preferably allantibodies contained, bind to different epitopes on the RABV G protein.

Preferably, the (at least) two antibodies according to the presentinvention are present in the pharmaceutical composition at equimolaramounts, preferably as an equimolar mixture.

The combination of two such antibodies according to the presentinvention, which bind to different epitopes of the RABV G proteinrepresents a treatment with an unprecedented breadth of reactivity andwith reduced risk of escape mutant selection. In particular, acombination of two or more monoclonal antibodies according to thepresent invention, whereby the antibodies bind to a different epitopesor sites on the RABV G protein, increases the protective effect andprevents the escape of resistant variants of the virus.

Preferably, compositions can include two or more (e.g., 2, 3, 4, 5 etc.)antibodies of the invention to provide an additive or synergistictherapeutic effect. The term “synergy” is used to describe a combinedeffect of two or more active agents that is greater than the sum of theindividual effects of each respective active agent. Thus, where thecombined effect of two or more agents results in “synergisticinhibition” of an activity or process, it is intended that theinhibition of the activity or process is greater than the sum of theinhibitory effects of each respective active agent. The term“synergistic therapeutic effect” refers to a therapeutic effect observedwith a combination of two or more therapies wherein the therapeuticeffect (as measured by any of a number of parameters) is greater thanthe sum of the individual therapeutic effects observed with therespective individual therapies.

In another embodiment, the composition may comprise one or more (e.g.,2, 3, etc.) antibodies of the invention and one or more (e.g., 2, 3,etc.) additional antibodies against a RABV and/or a non-RABV lyssavirus.Further, the administration of antibodies of the invention together withantibodies specific to other pathogens are within the scope of theinvention. The antibodies of the invention can be administered eithercombined/simultaneously or at separate times from antibodies of specificto pathogens other than a RABV and/or a non-RABV lyssavirus.

In another embodiment, the invention provides a pharmaceuticalcomposition comprising two or more antibodies, wherein the firstantibody is an antibody of the invention as described herein and thesecond antibody is specific for a different pathogen that may haveco-infected the subject to whom the pharmaceutical composition is beingadministered.

In a particularly preferred embodiment, the pharmaceutical compositionaccording to the present invention is administered in combination with arabies vaccine (active immunization), in particular in post-exposureprophylaxis. Currently available rabies vaccines include the most widelyused but highly risk-prone nerve tissue vaccines, or the safer but morecostly cell culture and embryonated egg vaccines (CCEEVs). In Germanye.g. only two anti-rabies vaccines are on the market, Rabipur® and“Tollwut-Impfstoff (human diploid cell [HDC]) inaktiviert”. Thesevaccines contain inactivated rabies virus. Both vaccines are recommendedfor pre- and postexposure use. Another example of a rabies vaccine isImovax (Sanofi-Pasteur), which is a commercial inactivated human diploidcell vaccine. Rabies vaccines are in general administered according tothe information of the manufacturer, whereby a typical post-exposureprophylaxis protocol includes administration of the vaccine at days 0,3, 7, 14 and 28 after infection. The pharmaceutical compositionaccording to the invention (passive immunization) is preferablyadministered only once, preferably simultaneously or as soon as possibleafter start of the vaccination. Preferably, the pharmaceuticalcomposition according to the invention is administered at a site distantfrom the vaccine.

Examples of antibodies of the invention specific for, and thatneutralize RABV and/or non-RABV lyssaviruses include, but are notlimited to, RVA122, RVA144, RVB185, RVB492, RVC3, RVC20, RVC21, RVC38,RVC44, RVC58, RVC68, and RVC111.

The combination, in particular an equimolar combination, of antibodiesaccording to gRVC20 and gRVC58, more preferably the antibodies RVC20 andRVC58, is particularly preferred. RVC20 binds to antigenic site I ofRABV G protein and RVC 58 binds to an epitope on the glycoprotein G ofRABV, which at least partially overlaps with antigenic site III on theglycoprotein G of RABV. Thus, a pharmaceutical composition comprisingthe antibodies RVC58 and RVC20 or an antigen binding fragment thereof,preferably in equimolar amounts, and a pharmaceutically acceptablecarrier is preferred.

Moreover, a pharmaceutical composition comprising the antibody accordingto gRVA122 or an antigen binding fragment thereof, and apharmaceutically acceptable carrier is also preferred. A pharmaceuticalcomposition comprising the antibody according to gRVA144 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier isalso preferred. A pharmaceutical composition comprising the antibodyaccording to gRVB185 or an antigen binding fragment thereof, and apharmaceutically acceptable carrier is also preferred. A pharmaceuticalcomposition comprising the antibody according to gRVB492 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier isalso preferred. A pharmaceutical composition comprising the antibodyaccording to gRVC3 or an antigen binding fragment thereof, and apharmaceutically acceptable carrier is also preferred. A pharmaceuticalcomposition comprising the antibody according to gRVC20 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier isalso preferred. A pharmaceutical composition comprising the antibodyaccording to gRVC21 or an antigen binding fragment thereof, and apharmaceutically acceptable carrier is also preferred. A pharmaceuticalcomposition comprising the antibody according to gRVC38 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier isalso preferred. A pharmaceutical composition comprising the antibodyaccording to gRVC44 or an antigen binding fragment thereof, and apharmaceutically acceptable carrier is also preferred. A pharmaceuticalcomposition comprising the antibody according to gRVC58 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier isalso preferred. A pharmaceutical composition comprising the antibodyaccording to gRVC68 or an antigen binding fragment thereof, and apharmaceutically acceptable carrier is also preferred. A pharmaceuticalcomposition comprising the antibody according to gRVC111 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier isalso preferred.

In addition, a pharmaceutical composition comprising the antibody RVA122or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier is also preferred. A pharmaceutical compositioncomprising the antibody RVA144 or an antigen binding fragment thereof,and a pharmaceutically acceptable carrier is also preferred. Apharmaceutical composition comprising the antibody RVB185 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier isalso preferred. A pharmaceutical composition comprising the antibodyRVB492 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier is also preferred. A pharmaceutical compositioncomprising the antibody RVC3 or an antigen binding fragment thereof, anda pharmaceutically acceptable carrier is also preferred. Apharmaceutical composition comprising the antibody RVC20 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier isalso preferred. A pharmaceutical composition comprising the antibodyRVC21 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier is also preferred. A pharmaceutical compositioncomprising the antibody RVC38 or an antigen binding fragment thereof,and a pharmaceutically acceptable carrier is also preferred. Apharmaceutical composition comprising the antibody RVC44 or an antigenbinding fragment thereof, and a pharmaceutically acceptable carrier isalso preferred. A pharmaceutical composition comprising the antibodyRVC58 or an antigen binding fragment thereof, and a pharmaceuticallyacceptable carrier is also preferred. A pharmaceutical compositioncomprising the antibody RVC68 or an antigen binding fragment thereof,and a pharmaceutically acceptable carrier is also preferred. Apharmaceutical composition comprising the antibody RVC111 variant 3 oran antigen binding fragment thereof, and a pharmaceutically acceptablecarrier is also preferred.

Antibodies may be administered to those subjects who have previouslyshown no response, i.e., have been shown to be refractive to treatmentfor RABV and/or non-RABV lyssavirus infection. Such treatment mayinclude previous treatment with an anti-viral agent. This may be due to,for example, infection with an anti-viral resistant strain of RABVand/or non-RABV lyssaviruses.

In one embodiment, a composition of the invention may include antibodiesof the invention, wherein the antibodies may make up at least 50% byweight (e.g., 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more)of the total protein in the composition. In such a composition, theantibodies are in purified form.

The invention provides a method of preparing a pharmaceuticalcomposition comprising the steps of: (i) preparing an antibody of theinvention; and (ii) admixing the purified antibody with one or morepharmaceutically-acceptable carriers.

In another embodiment, a method of preparing a pharmaceuticalcomposition comprises the step of: admixing an antibody with one or morepharmaceutically-acceptable carriers, wherein the antibody is amonoclonal antibody that was obtained from a transformed B cell or acultured plasma cell of the invention. Thus the procedures for firstobtaining the monoclonal antibody and then preparing the pharmaceuticalcan be performed at very different times by different people indifferent places (e.g., in different countries).

As an alternative to delivering antibodies or B cells for therapeuticpurposes, it is possible to deliver nucleic acid (typically DNA) thatencodes the monoclonal antibody (or active fragment thereof) of interestderived from the B cell or the cultured plasma cells to a subject, suchthat the nucleic acid can be expressed in the subject in situ to providea desired therapeutic effect. Suitable gene therapy and nucleic aciddelivery vectors are known in the art.

Compositions of the invention may be immunogenic compositions, and insome embodiments may be vaccine compositions comprising an antigencomprising an epitope recognized by an antibody of the invention or anantigen binding fragment thereof, in particular pharmaceuticalcompositions of the invention, which comprise an immunogenic polypeptideaccording to the invention. Such “vaccines” according to the inventionmay either be prophylactic (i.e., prevent infection) or therapeutic(i.e., treat or ameliorate infection). Such vaccine compositions of theinvention may elicit both a cell mediated immune response as well as ahumoral immune response in order to effectively address RABV andnon-RABV lyssavirus infection. This immune response may induce longlasting (e.g., neutralizing) antibodies and a cell mediated immunitythat can quickly respond upon exposure to RABV and/or non-RABVlyssavirus.

Compositions may include an antimicrobial, particularly if packaged in amultiple dose format. They may comprise detergent e.g., a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g., less than 0.01%. Compositions may also include sodium salts(e.g., sodium chloride) to give tonicity. A concentration of 10±2 mg/mlNaCl is typical.

Further, compositions may comprise a sugar alcohol (e.g., mannitol) or adisaccharide (e.g., sucrose or trehalose) e.g., at around 15-30 mg/ml(e.g., 25 mg/ml), particularly if they are to be lyophilized or if theyinclude material which has been reconstituted from lyophilized material.The pH of a composition for lyophilisation may be adjusted to between 5and 8, or between 5.5 and 7, or around 6.1 prior to lyophilisation.

The compositions of the invention may also comprise one or moreimmunoregulatory agents. In one embodiment, one or more of theimmunoregulatory agents include(s) an adjuvant.

Kit of Parts

In a further aspect, the present invention provides a kit of partscomprising at least one antibody, or antigen binding fragment thereof,according to the present invention as described herein, at least onenucleic acid according to the present invention as described herein, atleast one vector according to the present invention as described herein,at least one cell according to the present invention as describedherein, at least one immunogenic polypeptide according to the presentinvention as described herein, and/or at least one pharmaceuticalcomposition according to the present invention as described herein.

Preferably, such a kit of parts comprises at least two differentantibodies, or antigen binding fragments thereof, according to thepresent invention as described herein, wherein the antibodies, or theantigen binding fragments thereof, specifically bind to differentepitopes on the glycoprotein G of RABV. Such a kit of parts isparticularly useful for the combination of two antibodies according tothe present invention as described herein. The at least two antibodiesmay be present in the kit of parts as separate entities or combined,e.g. as a mixture, for example if both antibodies are contained in thesame pharmaceutical composition. Preferably, the at least two differentantibodies are separate entities in the kit of parts, which may be mixedby the user if needed. It is also preferred that the at least twodifferent antibodies are combined, e.g. as a mixture, for example ifboth antibodies are contained in the same pharmaceutical composition.

Preferably, in such a kit of parts one antibody of the at least twoantibodies binds to antigenic site I on the glycoprotein G of RABV andanother antibody of the at least two antibodies binds to antigenic siteIII on the glycoprotein G of RABV.

It is also preferred in such a kit of parts that at least one of the atleast two antibodies or antigen binding fragments thereof comprises aheavy chain CDRH3 comprising an amino acid sequence that is at least80%, preferably at least 90%, identical to SEQ ID NO: 95 or to SEQ IDNO: 167. More preferably, in such a kit of parts at least one of the atleast two antibodies or antigen binding fragments thereof comprises (i)heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 93-97 and 99 or to the amino acid sequences of SEQ ID NOs: 93-96and 98-99, respectively or (ii) heavy chain CDRH1, CDRH2, and CDRH3amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acidsequences that are at least 80%, preferably at least 90%, identical tothe amino acid sequences of SEQ ID NOs: 165-169 and 171 or to the aminoacid sequences of SEQ ID NOs: 165-168 and 170-171, respectively. Evenmore preferably, in such a kit of parts one of the at least twoantibodies or antigen binding fragments thereof comprises a heavy chainvariable region having at least 80%, preferably at least 90%, sequenceidentity to the amino acid sequence of SEQ ID NO: 107 or of SEQ ID NO:179.

Preferably, one of the at least two antibodies or antigen bindingfragments thereof comprises a heavy chain CDRH3 comprising an amino acidsequence that is at least 80%, preferably at least 90%, identical to SEQID NO: 95 and another of the at least two antibodies comprises a heavychain CDRH3 comprising an amino acid sequence that is at least 80%,preferably at least 90%, identical to SEQ ID NO: 167. More preferably,in such a kit of parts one of the at least two antibodies or antigenbinding fragments thereof comprises heavy chain CDRH1, CDRH2, and CDRH3amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acidsequences that are at least 80%, preferably at least 90%, identical tothe amino acid sequences of SEQ ID NOs: 93-97 and 99 or to the aminoacid sequences of SEQ ID NOs: 93-96 and 98-99, respectively, and anotherof the at least two antibodies comprises heavy chain CDRH1, CDRH2, andCDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences that are at least 80%, preferably at least 90%, identicalto the amino acid sequences of SEQ ID NOs: 165-169 and 171 or to theamino acid sequences of SEQ ID NOs: 165-168 and 170-171, respectively.Even more preferably, in such a kit of parts one of the at least twoantibodies or antigen binding fragments thereof is according to gRVC20and wherein another of the at least two antibodies is according togRVC58.

Medical Treatments and Uses

In a further aspect, the present invention provides the use of anantibody, or an antigen binding fragment thereof, according to thepresent invention, the nucleic acid according to the present invention,the vector according to the present invention, the cell according to thepresent invention, the immunogenic polypeptide according to the presentinvention, or the pharmaceutical composition according to the presentinvention in (i) prophylaxis, in particular post-exposure prophylaxis,treatment or attenuation of RABV and/or non-RABV lyssavirus infection;in (ii) vaccination against RABV and/or non-RABV lyssavirus infection;or in (iii) diagnosis of RABV and/or other lyssavirus infection.

Preferably, the antibodies and antibody fragments of the invention orderivatives and variants thereof may be used for the post-exposureprophylaxis and the treatment or attenuation of RABV and/or non-RABVlyssavirus infection, i.e., RABV infection or non-RABV lyssavirusinfection or co-infection with both RABV and non-RABV lyssavirus; forthe prevention of infection of RABV and/or non-RABV lyssavirus; or forthe diagnosis of RABV and/or non-RABV lyssavirus infection. Preferably,the antibody, or the antigen binding fragment thereof, according to thepresent invention may be used in the post-exposure prophylaxis,treatment or attenuation of infection of RABV and/or non-RABVlyssavirus.

A combination of two or more antibodies according to the presentinvention, e.g. a combination of 2, 3, 4, 5, 6, 7 etc. antibodiesaccording to the present invention, is particularly preferred for theuse in (i) prophylaxis, in particular post-exposure prophylaxis,treatment or attenuation of RABV and/or non-RABV lyssavirus infection;and in (ii) vaccination against RABV and/or non-RABV lyssavirusinfection. Such a combination may be for example a combination therapyas described below. Moreover, administration of the two or moreantibodies in equimolar amounts is preferred. In particular acombination of two antibodies comprising the six CDR regions accordingto RVC20 and RVC58, respectively, is preferred, a combination of twoantibodies according to gRVC20 and gRVC58 is more preferred, and acombination of the antibodies RVC20 and RVC58 is particularly preferred.

Moreover, in such a combination of two or more antibodies it ispreferred that at least two, preferably more than two, more preferablyall antibodies combined, bind to different epitopes on the RABV Gprotein as described above. For example, a first antibody in thecombination may specifically bind to antigenic site I on the RABV Gprotein and a second antibody may specifically bind to an epitope on theglycoprotein G of RABV, which at least partially overlaps with antigenicsite III on the glycoprotein G of RABV. The antigenic sites of theexemplary antibodies according to the present invention are outlined inExample 3. Moreover, whether two or more antibodies bind to the same ordifferent epitopes on the RABV G protein may be easily determined by theperson skilled in the art, for example by use of any competition study,whereby an example of a competition study is shown in Example 3. Aparticularly preferred example of a combination of two or moreantibodies according to the present invention is a combination ofantibodies according to gRVC20 and gRVC58, preferably RVC20 and RVC58.Moreover, a combination of two or more antibodies according to thepresent invention may for example include any combination of two or moreantibodies according to gRVA122, gRVA144, gRVB185, gRVB492, gRVC3,gRVC20, gRVC21, gRVC38, gRVC44, gRVC58, gRVC68, and gRVC111, preferablyany combination of two or more of RVA122, RVA144, RVB185, RVB492, RVC3,RVC20, RVC21, RVC38, RVC44, RVC58, RVC68, and RVC111.

Preferably, the two or more antibodies according to the presentinvention are administered in combination at equimolar amounts.

A “combination of (two or more) antibodies” as used herein refers to anycombination, for example the two or more antibodies may be contained inone pharmaceutical composition, or, preferably, the two or moreantibodies are administered as combination therapy, in particular theymay be administered separately from each other, e.g. in separateantibody preparations, for example in separate pharmaceuticalcompositions. This means, that in the combination preferably at leastone of the combined antibodies, more preferably 2, 3, 4, 5, or 6 of thecombined antibodies, more preferably every of the combined antibodiesis/are administered separately. In the combination therapy, theantibodies of the invention can be administered eithercombined/simultaneously ed/simultaneously or consecutively, i.e. oneantibody after the other.

In another embodiment, the combination may comprise one or more (e.g.,2, 3, etc.) antibodies of the invention and one or more (e.g., 2, 3,etc.) additional antibodies against a RABV and/or a non-RABV lyssavirus.Further, the administration of antibodies of the invention together withantibodies specific to other pathogens are within the scope of theinvention.

The antibody, or the antigen binding fragment thereof, the nucleic acid,the vector, the cell, the immunogenic polypeptide, or the pharmaceuticalcomposition according to the present invention may be provided for useas a medicament for (i) prophylaxis, in particular post-exposureprophylaxis, treatment or attenuation of RABV and/or non-RABV lyssavirusinfection; (ii) vaccination against RABV and/or non-RABV lyssavirusinfection; or (iii) diagnosis of RABV and/or other lyssavirus infection.

Within the scope of the invention are several forms and routes ofadministration of the antibody, or the antigen binding fragment thereof,the nucleic acid, the vector, the cell, the immunogenic polypeptide, orthe pharmaceutical composition, as described above, in respect to thepharmaceutical composition. This applies also in the context of the useof the antibody, or the antigen binding fragment thereof, the nucleicacid, the vector, the cell, the immunogenic polypeptide as describedherein, in particular regarding preferred forms and routes ofadministration.

Methods of diagnosis may include contacting an antibody or an antibodyfragment with a sample. Such samples may be tissue samples taken from,for example, nasal passages, sinus cavities, salivary glands, lung,liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart,ovaries, pituitary, adrenals, thyroid, brain or skin. The methods ofdiagnosis may also include the detection of an antigen/antibody complex.

The invention therefore provides (i) an antibody, an antibody fragment,or variants and derivatives thereof according to the invention, (ii) animmortalized B cell clone according to the invention, (iii) an epitopecapable of binding an antibody of the invention or (iv) a ligand,preferably an antibody, capable of binding an epitope that binds anantibody of the invention for use in therapy.

Preferably, the antibody, or the antigen binding fragment thereof,according to the present invention, the nucleic acid according to thepresent invention, the vector according to the present invention, thecell according to the present invention, the immunogenic polypeptideaccording to the present invention, or the pharmaceutical compositionaccording to the present invention are used in post-exposureprophylaxis, treatment or attenuation of RABV and/or non-RABV lyssavirusinfection, wherein the antibody, or the antigen binding fragmentthereof, the nucleic acid, the vector, the cell, or the pharmaceuticalcomposition is administered up to seven days, preferably up to fivedays, after infection.

The term “post-exposure prophylaxis” as used herein refers to atreatment protocol, which starts after exposure to the virus and beforethe first symptoms of rabies are detectable. However, since there iscurrently no real treatment, i.e. after start of the symptoms, forrabies, post-exposure prophylaxis may also be applied after the firstsymptoms of rabies are detectable, however, conventional post-exposureprophylaxis is known to be almost not effective at such a late timepoint.

In general, post-exposure prophylaxis is started as soon as possibleafter exposure or suspected exposure to the virus, preferably within afew hours until up to 24 hours or up to 48 hours after exposure. In thislimited time window, post-exposure prophylaxis is known to be mosteffective.

However, the antibodies according to the present invention are alsoeffective when administered at a later time point, presumably even afterstart of the first symptoms. Therefore, the antibodies according to thepresent invention considerably enlarge the time window for starting thepost-exposure prophylaxis, treatment or attenuation of RABV and/ornon-RABV lyssavirus infection. Preferably, the antibody, or the antigenbinding fragment thereof, the nucleic acid, the vector, the cell, or thepharmaceutical composition according to the present invention isadministered at least up to seven days, preferably at least up to fivedays, after infection.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention, the immunogenic polypeptideaccording to the present invention, or the pharmaceutical compositionaccording to the present invention is used in post-exposure prophylaxis,treatment or attenuation of RABV and/or non-RABV lyssavirus infection,wherein the antibody, or the antigen binding fragment thereof, thenucleic acid, the vector, the cell, or the pharmaceutical composition isadministered in combination with a vaccine, preferably a rabies vaccine,an antiviral, preferably ribavirin, interferon-alpha and/or ketamine.

As described above, currently available rabies vaccines include the mostwidely used but highly risk-prone nerve tissue vaccines, or the saferbut more costly cell culture and embryonated egg vaccines (CCEEVs). InGermany e.g. only two anti-rabies vaccines are on the market, Rabipur®and “Tollwut-Impfstoff (human diploid cell [HDC]) inaktiviert”. Thesevaccines contain inactivated rabies virus. Both vaccines are recommendedfor pre- and postexposure use. Another example of a rabies vaccine isImovax (Sanofi-Pasteur), which is a commercial inactivated human diploidcell vaccine. Rabies vaccines are in general administered according tothe information of the manufacturer, whereby a typical post-exposureprophylaxis protocol includes administration of the vaccine at days 0,3, 7, 14 and 28 after infection.

An antiviral refers to a class of medication used specifically fortreating viral infections. Like antibiotics for bacteria, specificantivirals are used for specific viruses. Unlike most antibiotics,antiviral drugs do not destroy their target pathogen; instead theyinhibit their development. Particularly preferred is the antiviralribavirin.

In a preferred embodiment, the antibody, or the antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention, the immunogenic polypeptideaccording to the present invention, or the pharmaceutical compositionaccording to the present invention are used in post-exposureprophylaxis, treatment or attenuation of RABV and/or non-RABV lyssavirusinfection, wherein the antibody, or the antigen binding fragmentthereof, the nucleic acid, the vector, the cell, or the pharmaceuticalcomposition is administered in a standard PEP scheme, preferably incombination with a vaccine, preferably in the first treatment of thestandard PEP scheme only.

A “standard PEP scheme” typically refers to the post-exposureprophylaxis scheme as recommended by the WHO (cf.who.int/rabies/human/WHO_strategy_prepost_exposure/en/index1.html#,retrieved at Nov. 12, 2014), whereby the antibodies according to theinvention replace the RIGs, i.e. HRIG or ERIG. Namely, a post-exposurevaccination is started as soon as possible after exposure with a rabiesvaccine as described herein, which follows the protocol of themanufacturer, typically at least two injections. For example, a standardprotocol includes injections of the vaccine at days 0, 3, 7, 14, and 28after exposure. Concomittantly to the first injection, or as soon aspossible afterwards, the only and single dose of the antibody isadministered.

When used in post-exposure prophylaxis, treatment or attenuation of RABVand/or non-RABV lyssavirus infection, preferably the antibody, or theantigen binding fragment thereof, according to the present invention ispreferably administered at a dose of 0.005 to 100 mg/kg, preferably at adose of 0.0075 to 50 mg/kg, more preferably at a dose of 0.01 to 10mg/kg, even more preferably at a dose of 0.01 to 1 mg/kg, andparticularly preferably at a dose of 0.01 to 0.1 mg/kg. Such a dose isparticularly preferred in a standard PEP scheme as described above.

It is also preferred that the antibody, or the antigen binding fragmentthereof, according to the present invention, the nucleic acid accordingto the present invention, the vector according to the present invention,the cell according to the present invention, the immunogenic polypeptideaccording to the present invention, or the pharmaceutical compositionaccording to the present invention for use in post-exposure prophylaxis,treatment or attenuation of RABV and/or non-RABV lyssavirus infection,is administered from 1 to 6 days, preferably from 2 to 5 days, afterinfection.

In another preferred embodiment, which does not refer to the standardPEP scheme, the antibody, or the antigen binding fragment thereof,according to the present invention, for use in post-exposureprophylaxis, treatment or attenuation of RABV and/or non-RABV lyssavirusinfection, is administered without concomitant and/or subsequentadministration of a vaccine.

In addition to administration in combination with a vaccine, e.g. in astandard PEP scheme, the antibodies according to the present inventionare also effective when administered without a vaccine, for example astreatment of rabies, e.g. if administered more than one or two daysafter exposure.

Moreover, it is preferred that in the antibody, or the antigen bindingfragment thereof, according to the present invention or in thepharmaceutical composition according to any of claims 21 to 24 for usein post-exposure prophylaxis, treatment or attenuation of RABV and/ornon-RABV lyssavirus infection, that the antibody, or the antigen bindingfragment thereof, is administered at a dose of 0.01 to 100 mg/kg,preferably at a dose of 0.1 to 75 mg/kg, more preferably at a dose of 1to 60 mg/kg, and even more preferably at a dose of 10 to 50 mg/kg. Such“higher” doses are in particular preferred if the exposure was severeand/or if treatment is initiated later than one or two days afterexposure.

The invention also provides a method of treating a subject comprisingadministering to the subject an antibody, an antibody fragment, orvariants and derivatives thereof according to the invention, or, aligand, preferably an antibody, capable of binding an epitope that bindsan antibody of the invention. In one embodiment, the method results inreduced RABV and/or non-RABV lyssavirus infection in the subject. Inanother embodiment, the method prevents, reduces the risk or delays RABVand/or non-RABV lyssavirus infection in the subject.

In particular, the present invention provides a method of preventingand/or treating a RABV and/or non-RABV lyssavirus infection in asubject, wherein the method comprises administering to a subject in needthereof the antibody, or the antigen binding fragment thereof, accordingto the present invention as described herein, the nucleic acid accordingto according to the present invention as described herein, the vectoraccording to according to the present invention as described herein, thecell according to according to the present invention as describedherein, the immunogenic polypeptide according to according to thepresent invention as described herein, and/or the pharmaceuticalcomposition according to according to the present invention as describedherein. Such a method preferably comprises post-exposure prophylaxis asdescribed herein. It is also preferred that such a method comprisesvaccination against RABV and/or non-RABV lyssavirus infection.

The present invention also provides a method of diagnosing a RABV and/ornon-RABV lyssavirus infection in a subject, wherein the method comprisesadministering to a subject in need thereof the antibody, or the antigenbinding fragment thereof, according to the present invention asdescribed herein, the nucleic acid according to the present invention asdescribed herein, the vector according to the present invention asdescribed herein, the cell according to the present invention asdescribed herein, the immunogenic polypeptide according to the presentinvention as described herein, and/or the pharmaceutical compositionaccording to the present invention as described herein.

Preferably, in the above described methods according to the presentinvention at least two antibodies, or antigen binding fragments thereof,according to the present invention as described herein are administeredto the subject, which antibodies, or antigen binding fragments thereof,specifically bind to different epitopes on the glycoprotein G of RABV.Preferably one antibody of the at least two antibodies binds toantigenic site I on the glycoprotein G of RABV and another antibody ofthe at least two antibodies binds to antigenic site III on theglycoprotein G of RABV. Preferably at least one of the at least twoantibodies or antigen binding fragments thereof comprises a heavy chainCDRH3 comprising an amino acid sequence that is at least 80%, preferablyat least 90%, identical to SEQ ID NO: 95 or to SEQ ID NO: 167. Morepreferably, at least one of the at least two antibodies or antigenbinding fragments thereof comprises (i) heavy chain CDRH1, CDRH2, andCDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences that are at least 80%, preferably at least 90%, identicalto the amino acid sequences of SEQ ID NOs: 93-97 and 99 or to the aminoacid sequences of SEQ ID NOs: 93-96 and 98-99, respectively or (ii)heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 165-169 and 171 or to the amino acid sequences of SEQ ID NOs:165-168 and 170-171, respectively. Even more preferably, one of the atleast two antibodies or antigen binding fragments thereof comprises aheavy chain CDRH3 comprising an amino acid sequence that is at least80%, preferably at least 90%, identical to SEQ ID NO: 95 and another ofthe at least two antibodies comprises a heavy chain CDRH3 comprising anamino acid sequence that is at least 80%, preferably at least 90%,identical to SEQ ID NO: 167. Particularly preferably, one of the atleast two antibodies or antigen binding fragments thereof comprisesheavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences that are at least 80%,preferably at least 90%, identical to the amino acid sequences of SEQ IDNOs: 93-97 and 99 or to the amino acid sequences of SEQ ID NOs: 93-96and 98-99, respectively, and another of the at least two antibodiescomprises heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences andlight chain CDRL1, CDRL2, and CDRL3 amino acid sequences that are atleast 80%, preferably at least 90%, identical to the amino acidsequences of SEQ ID NOs: 165-169 and 171 or to the amino acid sequencesof SEQ ID NOs: 165-168 and 170-171, respectively.

The invention also provides the use of (i) an antibody, an antibodyfragment, or variants and derivatives thereof according to theinvention, (ii) an immortalized B cell clone according to the invention,(iii) an epitope capable of binding an antibody of the invention, (iv) aligand, preferably an antibody, that binds to an epitope capable ofbinding an antibody of the invention, or (v) a pharmaceuticalcomposition of the invention in (i) the manufacture of a medicament forthe treatment or attenuation of infection by RABV and/or non-RABVlyssavirus (ii) a vaccine, or (iii) diagnosis of RABV and/or non-RABVlyssavirus infection.

The invention provides a composition of the invention for use as amedicament for the prevention or treatment of RABV and/or non-RABVlyssavirus infection. It also provides the use of an antibody of theinvention and/or a protein comprising an epitope to which such anantibody binds in the manufacture of a medicament for treatment of asubject and/or diagnosis in a subject. It also provides a method fortreating a subject, comprising the step of administering to the subjecta composition of the invention. In some embodiments the subject may be ahuman. One way of checking efficacy of therapeutic treatment involvesmonitoring disease symptoms after administration of the composition ofthe invention. Treatment can be a single dose schedule or a multipledose schedule.

In one embodiment, an antibody, antibody fragment, immortalized B cellclone, epitope or composition according to the invention is administeredto a subject in need of such treatment. Such a subject includes, but isnot limited to, one who is particularly at risk of or susceptible toRABV and/or non-RABV lyssavirus infection.

Antibodies and fragments thereof as described in the present inventionmay also be used in a kit for the diagnosis of RABV and/or non-RABVlyssavirus infection. Further, epitopes capable of binding an antibodyof the invention may be used in a kit for monitoring the efficacy ofvaccination procedures by detecting the presence of protective anti-RABVor anti-non-RABV lyssavirus antibodies. Antibodies, antibody fragment,or variants and derivatives thereof, as described in the presentinvention may also be used in a kit for monitoring vaccine manufacturewith the desired immunogenicity.

Antibodies and fragments thereof as described in the present inventionmay also be used for monitoring the quality of anti-RABV oranti-non-RABV lyssavirus vaccines by checking that the antigen of saidvaccine contains the specific epitope in the correct conformation.

The invention also provides an epitope that specifically binds to anantibody of the invention or an antigen binding fragment thereof, foruse (i) in therapy, (ii) in the manufacture of a medicament for thetreatment or attenuation of RABV and/or non-RABV lyssavirus infection,(iii) as a vaccine, or (iv) in screening for ligands able to neutralizeRABV and/or non-RABV lyssavirus infection.

The invention also provides a method of preparing a pharmaceutical,comprising the step of admixing a monoclonal antibody with one or morepharmaceutically-acceptable carriers, wherein the monoclonal antibody isa monoclonal antibody that was obtained from a transfected host cell ofthe invention. Thus the procedures for first obtaining the monoclonalantibody (e.g., expressing it and/or purifying it) and then admixing itwith the pharmaceutical carrier(s) can be performed at very differenttimes by different people in different places (e.g., in differentcountries).

Starting with a transformed B cell or a cultured plasma cell of theinvention, various steps of culturing, sub-culturing, cloning,sub-cloning, sequencing, nucleic acid preparation etc. can be performedin order to perpetuate the antibody expressed by the transformed B cellor the cultured plasma cell, with optional optimization at each step. Inone embodiment, the above methods further comprise techniques ofoptimization (e.g., affinity maturation or optimization) applied to thenucleic acids encoding the antibody. The invention encompasses allcells, nucleic acids, vectors, sequences, antibodies etc. used andprepared during such steps.

In all these methods, the nucleic acid used in the expression host maybe manipulated to insert, delete or alter certain nucleic acidsequences. Changes from such manipulation include, but are not limitedto, changes to introduce restriction sites, to amend codon usage, to addor optimize transcription and/or translation regulatory sequences, etc.It is also possible to change the nucleic acid to alter the encodedamino acids. For example, it may be useful to introduce one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions,deletions and/or insertions into the antibody's amino acid sequence.Such point mutations can modify effector functions, antigen-bindingaffinity, post-translational modifications, immunogenicity, etc., canintroduce amino acids for the attachment of covalent groups (e.g.,labels) or can introduce tags (e.g., for purification purposes).Mutations can be introduced in specific sites or can be introduced atrandom, followed by selection (e.g., molecular evolution). For instance,one or more nucleic acids encoding any of the CDR regions, heavy chainvariable regions or light chain variable regions of antibodies of theinvention can be randomly or directionally mutated to introducedifferent properties in the encoded amino acids. Such changes can be theresult of an iterative process wherein initial changes are retained andnew changes at other nucleotide positions are introduced. Further,changes achieved in independent steps may be combined. Differentproperties introduced into the encoded amino acids may include, but arenot limited to, enhanced affinity.

The following Figures, Sequences and Examples are intended to illustratethe invention further. They are not intended to limit the subject matterof the invention thereto.

DESCRIPTION OF FIGURES

FIG. 1 shows a summary of the characteristics of the RABV and non-RABVlyssavirus isolates referred to herein. This includes isolate name,viral species and phylogroup (as shown in Table 1 for the non-RABVlyssavirus isolates) as well as host species, country and year oforigin, lineage and the GenBank accession number of the amino acidand/or nucleotide sequence of the glycoprotein G of that isolate, ifavailable.

FIG. 2 shows the results of RABV G-protein binding (A) andneutralization (B) by a panel of 90 and 29 plasma samples from RABVvaccines, respectively. Black symbols indicate HRIG (Berirab®), greysymbols indicate the 4 donors selected for the memory B cellinterrogation.

FIG. 3 shows a summary of all the genetic and functional characteristicsof the panel of 21 isolated human RABV neutralizing antibodies. Shownare VH, VL and VK usage, the percentage of nucleotide identity to thecorresponding germline gene, the neutralization potency on CVS-11 RABVpseudoviruses (pp) expressed as the concentration of IgG in ng/ml ableto neutralize 90% of viral infectivity (IC₉₀) and the reactivity of theantibodies in western blot (WB) under non-reducing or reducingconditions.

FIG. 4 shows the results of a monoclonal antibody cross-competitionmatrix performed by ELISA on the 21 isolated antibodies and tworeference antibodies of known epitope specificity (CR57 and CR4098).Shown is the percentage of inhibition of binding of the biotinylatedantibodies shown in the upper row by the antibodies listed in the leftcolumn.

FIG. 5 shows the results of neutralization of 13 different lyssavirusspecies (22 viral isolates) tested as pseudoviruses by a selection of 12human monoclonal antibodies from the isolated panel as compared to thethree reference antibodies RAM, CR57 and CR4098 and the polyclonal humanimmunoglobulins (HRIG, Berirab®). Shown is the IC₉₀ value in ng/ml,whereby IC₉₀>10′000 ng/ml were scored as negative (values for Berirab®neutralization are scored as negative if IC₉₀>50′000 ng/ml).

FIG. 6 shows the results of neutralization of 8 different lyssavirusspecies (16 viral isolates) tested as infectious viruses by a selectionof 12 human monoclonal antibodies from the isolated panel as compared tothe three reference antibodies RAM, CR57 and CR4098 and the polyclonalhuman immunoglobulins (HRIG, Berirab®). Shown is the IC₅₀ value inng/ml, whereby IC₅₀>10′000 ng/ml were scored as negative (values forBerirab® neutralization are scored as negative if IC₅₀>50′000 ng/ml).

FIG. 7 shows the results of neutralization of 13 different lyssavirusspecies tested as pseudoviruses (A, 22 viral isolates) or viruses (B, 16viral isolates) by a selection of 12 monoclonal antibodies from theisolated panel as compared to the two reference CR57 and CR4098antibodies and the polyclonal human immunoglobulins (HRIG, Berirab®).

FIG. 8 shows a summary of the percentage of non-RABV lyssavirus isolates(n=32) (A) and phylogroup I non-RABV lyssavirus isolates (n=22) (B)neutralized with IC₅₀ (for viruses) or IC₉₀ (for pseudoviruses) below10000 ng/ml by RVC20, RVC58, RAB1, CR57, CR4098 monoclonal antibodies,or a combination of RVC20 with RVC58 or CR57 with CR4098. The list ofthe isolates (and their phylogroup) used for this analysis is shown inFIGS. 5 and 6 . N, number of isolates used in the calculation of theneutralized isolates. *, HRIG was scored as negative when IC₅₀ or IC₉₀was >50′000 ng/ml; **, RAM was tested against 26 non RABV-isolates and16 non-RABV phylogroup I isolates, respectively.

FIG. 9 shows the results of neutralization of RABV isolates tested aspseudoviruses (filled circles, n=2) or viruses (empty circles, n=24) bythe selected RVC20 and RVC58 antibodies from our panel and the tworeference CR57 and CR4098 antibodies.

FIG. 10 shows that RVC20 and RVC58 potently neutralize multi-lineageRABV isolates. (A) Neutralization of RABV isolates tested as forpseudotyped viruses (filled circles, n=8; shown are IC₉₀ values) or liveviruses (empty circles, n=27; shown are the IC₅₀ values) by the selectedRVC20 and RVC58 antibodies from our panel, the reference CR57, CR4098and RAB1 antibodies and HRIG. CVS-11 strain neutralization is shownusing live viruses (RFFIT assay, see FIG. 11 ). Dotted line indicate athreshold for neutralization above 1′000 ng/ml. Shown is the geometricmean value for each data set. The P value of a Wilcoxon matched-pairssigned rank test (****P<0.0001; ***P<0.001) is shown. (B) Phylogenetictree of 2215 G protein sequences retrieved from public databases.Highlighted with dots are the sequences of the RABV isolates tested inthis work (two G protein sequences, i.e. CV9.13 andMauritania/dog/2019-2006/V6235-2007, were not available and weretherefore not included in the tree) including those that were tested byFACS for binding (cf. FIG. 11 ).

FIG. 11 shows a summary of the 43 RABV isolates tested. Neutralizationactivity (IC₅₀ for viruses and IC₉₀ for pseudvoviruses in ng/ml) ofRVC20, RVC58, CR57, CR4098 and RAB1 monoclonal antibodies and HRIG asillustrated in FIG. 10 . RFFIT, rapid fluorescent focus inhibition test;FAVN, fluorescent-antibody virus neutralization test; PV,pseudovirus-based neutralization assay. *, viruses tested by FACS forbinding to G protein transfectants.

FIG. 12 shows the characteristics of 26 selected RABV isolates tested asviruses or pseudoviruses as well as the neutralization activity in ng/mlof RVC20, RVC58, CR57, CR4098 monoclonal antibodies and HRIG asillustrated in FIGS. 9 and 10 . RFFIT, rapid fluorescent focusinhibition test; FAVN, fluorescent-antibody virus neutralization test;PV, pseudovirus-based neutralization assay. Shown are IC₅₀ for the FAVNand RFFIT results and IC₉₀ for the PV results.

FIG. 13 shows the characteristics of 28 selected non-RABV lyssavirusisolates tested as viruses or pseudoviruses as well as theneutralization activity in ng/ml of RVC20, RVC58, CR57, CR4098monoclonal antibodies and HRIG as illustrated in FIGS. 5, 6 and 7 .RFFIT, rapid fluorescent focus inhibition test; FAVN,fluorescent-antibody virus neutralization test; PV, pseudovirus-basedneutralization assay. Shown are IC₅₀ for the FAVN and RFFIT results andIC₉₀ for the PV results.

FIG. 14 shows the results of neutralization of CVS-11 and differentCVS-11 mutants by the panel of 12 selected monoclonal antibodiesaccording to the invention and the reference antibodies RAB1, CR57 andCR4098. Black cells indicate full neutralization, grey cells partialneutralization and white cells no neutralization. Nd, not tested.

FIG. 15 shows that RVC20 and RVC58 target highly conserved epitopes inantigenic sites I and III. Level of amino acid residue conservation inantigenic sites I and III as calculated by the analysis of the G-proteinsequences from 2566 RABVs. Pie charts shows the detailed distribution ofamino acid usage at each position. Underlined residues indicate thatviruses carrying the corresponding residue in that position areneutralized by either RVC20 or RVC58. (A) Frequency of amino acidresidues in antigenic site I; (B) Frequency of amino acid residues inantigenic site III.

FIG. 16 shows an alignment of antigenic site sequences of the G proteinfrom all the tested and sequenced lyssaviruses. Shown are phylogroup Inon-RABV and phylogroup II-IV non-RABV lyssaviruses, respectively. Nd,not tested. (A) Antigenic site I with arrows in the first columnindicating a lack of neutralization by CR57 and arrows in the secondcolumn (most right column of panel A) indicating a lack ofneutralization by RVC20. (B) Antigenic site III with arrows in the firstcolumn indicating a lack of neutralization by CR4098, arrows in thesecond column indicating a lack of neutralization by RAB1 and arrows inthe third column (most right column of panel B) indicating a lack ofneutralization by RVC58. Dotted arrows indicate weak or partialneutralization.

FIG. 17 shows the percent survival in Syrian hamsters infected with RABVCVS-11 isolate and then left untreated or treated with the standard PEP(HRIG and vaccination) or with two different doses of a cocktail ofRVC20+RVC58 monoclonal antibodies (and vaccination). The RABV virus wasadministered intramuscularly (50 μl of 10^(5.7) TCID50/ml) in thegastrocnemius muscle of the hind left leg. The vaccine used is acommercial inactivated human diploid cell vaccine (Imovax;Sanofi-Pasteur) and was administered intramuscularly (0.05 ml) in the inthe gastrocnemius muscle of the hind right leg.

FIG. 18 shows the level of hamster IgG antibodies binding to G proteinas measured by ELISA (A), the level of hamsters sera neutralizingantibodies (B) and the levels of residuals human IgG antibodies (C) asmeasured on sera collected 42 days after immunization with RABV vaccinein unchallenged Syrian hamsters.

FIG. 19 shows the percent survival in Syrian hamsters infected with afield RABV virus isolated from the salivary glands of an infected fox(Italy/red fox/673/2011) and then left untreated, treated on day 1 withthe standard PEP (i.e. HRIG and vaccination) or treated either on day 1,5 or 9 after infection with a single dose of RVC58+RVC20 at 40 mg/kg.

FIG. 20 shows the RABV NP mRNA amounts as measured by RT-PCR onpost-mortem CNS samples (A) and the levels of hamster IgG antibodiesbinding to G protein as measured by ELISA (B) from the experiment shownin FIG. 18 . Asterisks indicate animals succumbing the infection,diamonds animals showing a permanent paralysis of the back leg that wassite of the viral challenge and open circles asymptomatic animals.

FIG. 21 shows the histologic analysis of brain, medulla oblongata andspinal cord tissues from two representative animals treated with RVC58and RVC20 on day 5 after infection (A) or left treated (B). Inparticular, the immunohistochemistry analysis was aimed to reveal thepresence of RABV N antigen to identify the pathognomonic inclusionbodies (Negri bodies).

FIG. 22 shows the amino acid sequences for the heavy and light chains ofantibody RVA122 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 23 shows the amino acid sequences for the heavy and light chains ofantibody RVA144 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 24 shows the amino acid sequences for the heavy and light chains ofantibody RVB185 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 25 shows the amino acid sequences for the heavy and light chains ofantibody RVB492 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 26 shows the amino acid sequences for the heavy and light chains ofantibody RVC3 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 27 shows the amino acid sequences for the heavy and light chains ofantibody RVC20 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 28 shows the amino acid sequences for the heavy and light chains ofantibody RVC21 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 29 shows the amino acid sequences for the heavy and light chains ofantibody RVC38 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 30 shows the amino acid sequences for the heavy and light chains ofantibody RVC44 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 31 shows the amino acid sequences for the heavy and light chains ofantibody RVC58 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 32 shows the amino acid sequences for the heavy and light chains ofantibody RVC6S as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

FIG. 33 shows the amino acid sequences for the heavy and light chains ofantibody RVC111 as well as the nucleic acid sequences that encode them.The sequences highlighted in bold are CDR regions (nucleotide or aa) andthe underlined residues are mutated residues as compared to the“germline” sequence.

EXAMPLES

Exemplary embodiments of the present invention are provided in thefollowing examples. The following examples are presented only by way ofillustration and to assist one of ordinary skill in using the invention.The examples are not intended in any way to otherwise limit the scope ofthe invention.

Example 1

Selection of Rabies Vaccines for the Isolation of Broadly NeutralizingAntibodies.

In order to isolate broadly neutralizing antibodies capable toneutralize RABV isolates but also non-RABV lyssaviruses, 90 plasmasamples from vaccines were screened for the presence of high titers ofantibodies binding to RABV G protein (CVS-11 strain) by ELISA (FIG. 2A)and selected 29 samples with the highest binding titers (EC₅₀>50) forfurther analysis. In particular the selected 29 plasma samples weretested for their ability to neutralize on a panel of 12 pseudotypedlyssaviruses including phylogroup I viruses RABV, DUVV, KHUV, EBLV1,ARAV, EBLV2, IRKV, ABLV, phylogroup II viruses LABV, SHIBV, MOKV andphylogroup III WCBV (FIG. 2B). Human rabies immunoglobulin (HRIG)Berirab® (Zydus Cadila) was included as a reference. As expected allsamples neutralized, albeit with variable titers, the homologous RABVCVS-11 isolate. The neutralization profile of the other lyssavirusspecies varied considerably in all donors tested where in a few casesall species were neutralized. Of note, HRIG (Berirab®) showed onlymodest activity against non-RABV phylogroup I species, andno-crossreactivity with phylogroup II and III viruses. This analysisallowed to select four vaccines as blood donors for the subsequentisolation and characterization of potent broadly neutralizingantibodies.

Example 2

Isolation and Characterization of Rabies Broadly NeutralizingAntibodies.

IgG+ memory B cells were isolated from cryopreserved PBMCs of the fourselected vaccines using CD22 microbeads (Miltenyi Biotec), followed bydepletion of cells carrying IgM, IgD and IgA by cell sorting. Memory Bcells from the four selected vaccines were then immortalized with EBV(Epstein Barr Virus) and CpG (CpG oligodeoxynucleotide 2006) in multiplereplicate wells as previously described (Traggiai, E. et al., Nat. Med.10, 871-875, 2004) and culture supernatants were then tested in aprimary screening using a 384-well based CSV-11 RABV pseudotypedneutralization assay (CVS-11 reference isolate, vaccine strain). Humanembryonic kidney 293T cells were used for production of the lentiviralpseudotypes (lyssavirus surrogates). Neutralisation assays wereundertaken on baby hamster kidney 21 cells clone 13 (BHK). In a 384-wellplate, CVS-11 pseudovirus that resulted in an output of 50-100×10⁴relative light units (RLU) was incubated with doubling dilutions of serafor 1 h at 37% (5% CO2) before the addition of 3′000 BHK-21 cells. Thesewere incubated for a further 48 h, after which supernatant was removedand 15 μl Steadylite reagent (Perkin Elmer) was added. Luciferaseactivity was detected 5 min later by reading the plates on a Synergymicroplate luminometer (BioTek) (Wright, E. et al., J Gen. Virol 89,2204-2213, 2008). Positive cultures were collected and expanded. Frompositive cultures the VH and VL sequences were retrieved by RT-PCR.RVC20 and RVC58 antibodies were cloned into human IgG1 and Ig kappa orIg lambda expression vectors (kindly provided by Michel Nussenzweig,Rockefeller University, New York, US) essentially as described (TillerT, Meffre E, Yurasov S, Tsuiji M, Nussenzweig M C, Wardemann H (2008)Efficient generation of monoclonal antibodies from single human B cellsby single cell RT-PCR and expression vector cloning. J Immunol Methods329: 112-124). Monoclonal antibodies were produced from EBV-immortalizedB cells or by transient transfection of 293 Freestyle cells(Invitrogen). Supernatants from B cells or transfected cells werecollected and IgG were affinity purified by Protein A or Protein Gchromatography (GE Healthcare) and desalted against PBS.

Five hundred human monoclonal antibodies were isolated for their abilityto neutralize RABV. Twenty-one human monoclonal antibodies were selectedfor their high neutralizing potency against CVS-11 RABV, with IC₉₀(concentration of antibody neutralizing 90% of viral infectivity)ranging from 0.01 to 317 ng/ml (FIG. 3 ), produced by EBV-immortalized Bcells and affinity purified by Protein A or Protein G (in the case ofIgG3) chromatography. As a reference HRIG and two other human monoclonalantibodies (CR57 and CR4098) that were developed up to Phase III toreplace RIGs (but recently failed due to the lack of neutralizingactivity against some circulating field RABV isolates) were also tested.In addition, all antibodies were then shown to bind to RABV G protein byELISA (CVS-11). To this end, a standard ELISA was used. Briefly, ELISAplates were coated with RABV G protein at 5 μg/ml, blocked with 10% FCSin PBS, incubated with sera or human antibodies and washed. Boundantibodies were detected by incubation with AP-conjugated goatanti-human IgG (Southern Biotech). Plates were then washed, substrate(p-NPP, Sigma) was added and plates were read at 405 nm. The relativeaffinities of sera binding or monoclonal antibody binding weredetermined by measuring the dilution of sera (ED50) or the concentrationof antibody (EC50) required to achieve 50% maximal binding atsaturation.

In order to understand whether the cognate epitope is conformational ornot the RABV G protein was run on a SDS-PAGE gel under reducing (RED) ornon-reducing (NR) conditions and probed by Western blot with all theisolated human monoclonal antibodies. With a few exceptions (RVB143,RVC44 and RVC68) all antibodies did not bind to RABV G protein underreducing conditions, thus suggesting that the epitope recognized isconformational (FIG. 3 ).

Example 3

Antibody Competition Studies: Determination of Antigenic Sites on RABV GProtein.

Competition studies were then performed to determine the spatialproximity of each of the conformational epitopes recognized by the allantibodies of the panel. The two reference antibodies CR57 and CR4098were previously shown to recognize G protein antigenic sites I and III(Bakker, A. B. H. et al., J Virol 79, 9062-9068, 2005; de Kruif, J. etal., Annu Rev Med 58, 359-368, 2007), respectively, and were thereforeused in this assay as probes to map the specificity of each antibody ofour panel. In particular, CR57, CR4098 and all 21 antibodies selectedwere purified and labeled with biotin and then tested by ELISA in a fullmatrix competition assay, in which unlabeled antibodies were incubatedfirst at a concentration of 10 μg/ml on RABV G protein coated plates,followed by the addition of biotinylated antibodies at a concentrationof 100 ng/ml (i.e. 100 fold less than the unlabeled antibody), whosebinding was revealed with alkaline-phosphatase conjugated streptavidin.Results shown in FIG. 4 and indicate the percentage of blocking ofbinding of the labeled antibodies in all possible combinations (i.e.21×21) and were used to cluster antibodies into 6 groups. Wheninterpreting competition results, it should be taken into account thatif two epitopes overlap, or the areas covered by the arms of the twoantibodies overlap, competition should be almost complete. Weakinhibitory or enhancing effects may reflect a decrease in affinity owingto steric or allosteric effects.

RVA125, RVC3, RVC20 and RVD74 were assigned to the antigenic site Igroup according to the competition with CR57 and to their reciprocalcompetitions. Of note, the binding of antigenic site I antibodies to Gprotein is enhanced by a subgroup of non-antigenic site-I antibodies.RVA122, RVA144, RVB492, RVC4, RVC69, RVC38 and RVC58 were assigned tothe antigenic site III according to the competition with CR4098 and totheir reciprocal competitions. RVC58 showed only a partial competitionwith CR4098 (i.e. 64%) as well as competition with non-antigenic site Iand III antibodies, thus suggesting that the RVC58 epitope might onlypartially overlap with antigenic site III. A third cluster composed byantibodies RVB181, RVC56, RVB185, RVC21, RVB161 and RVC111 was named111.2 since the binding of all these biotinylated antibodies was blockedby all antigenic site III antibodies but reciprocally all theseantibodies were not able to block the binding of several antigenic siteIII antibodies like CR4098, RVC4 and RVC69. In interpreting competitionresults, it should be taken into account that when two epitopes overlap,or when the areas covered by the arms of the two antibodies overlap,competition should be almost complete. Weak inhibitory or enhancingeffects may simply reflect a decrease in affinity owing to steric orallosteric effects. For this reason here we have defined a novel sitecalled 111.2, which is likely proximal to antigenic site III on the Gprotein. Following the same criteria three additional sites were definednamed A, B and C. The site A is defined by the unique antibody RVB686,whose binding compromises the binding of the majority of the labeledantibodies of the panel, but reciprocally the binding of the labeledRBV686 is not blocked by any antibody of the panel. These results mightsuggest that RVB686 binding induces an allosteric effect on the Gprotein that compromises the binding of most other antibodies. Site B isdefined by antibody RVC44, whose binding is not blocked by any otherantibody of the panel. Similarly, site C is defined by antibodies RVB143and RVC68, which also recognize a unique and distinct epitope ascompared to all the other antibodies. Of note, RVC44, RVB143 and RVC68are the only antibodies of this panel capable of binding by western blotto G protein under reducing conditions, suggesting that they recognize alinear epitope on RABV G protein.

Example 4

The Antibodies According to the Present Invention Potently NeutralizeRABV and Non-RABV Lyssaviruses.

Twelve of the 22 antibodies were selected for their potency and for therecognition of distinct sites on the RABV G protein for being tested,along with the reference antibodies CR57, CR4098, RAM and Berirab®(HRIG), against a large panel of lyssaviruses using pseudotyped (22isolates, as shown in FIG. 5 ) and infectious viruses (16 isolates, asshown in FIG. 6 ) covering RABV, DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV,ARAV, LBV, MOK, SHIBV, BBLV, WCBV and IKOV species (FIGS. 5, 6 and 7 ).

Production of Pseudotyped Viruses and Neutralization Assay.

Human embryonic kidney 293T clone 17 cells (HEK 293T/17; ATCC CRL-11268)were used for production of the lentiviral pseudotypes. Neutralisationassays were undertaken on BHK-21 cells clone 13 (ATCC CCL-10). In a384-well plate, pseudotyped virus that resulted in an output of50-100×10⁴ relative light units (RLU) was incubated with doublingdilutions of sera or antibodies for 1 h at 37% (5% CO2) before theaddition of 3′000 BHK-21 cells. These were incubated for a further 48hours, after which supernatant was removed and 15 μl Steadylite reagent(Perkin Elmer) was added. Luciferase activity was detected 5 min laterby reading the plates on a Synergy microplate luminometer (BioTek)(Wright et al. 2008). The reduction of infectivity was determined bycomparing the RLU in the presence and absence of antibodies andexpressed as percentage of neutralization. The neutralization potencyfor the monoclonal antibodies is here measured as IC₉₀, which wasdefined as the antibody concentration at which RLU were reduced 90%compared with virus control wells after subtraction of background RLU incell control wells (ID50 for the sera, i.e. the dilution of sera atwhich RLU were reduced 50%). ID₅₀ values for the sera correspond to thedilution at which RLU were reduced 50%.

Lyssavirus Cell-Adaptation and In Vitro Neutralization Assays.

Selected RABVs and non-RABV lyssaviruses were isolated on Neuro-2A (ATCCcat n. CCL-131), further cell adapted and working stocks produced andtitrated on BSR cells (a clone of BHK-21). Two protocols slightlymodified from Fluorescent Antibody Virus Neutralization (mFAVN) and fromRapid Fluorescent Foci Inhibition (mRFFIT) test (FAVN: Cliquet, F., etal., J. Immunol Methods 212, 79-87, 1998; RFFIT: Smith, J. S., et al.,Bull. World Health Organ. 48, 535-541, 1973, Warrell M J, Riddell A, YuL M, Phipps J, Diggle L, Bourhy H, Deeks J J, Fooks A R, Audry L,Brookes S M, et al (2008) A simplified 4-site economical intradermalpost-exposure rabies vaccine regimen: a randomised controlled comparisonwith standard methods. PLoS Negl Trop Dis 2: e224), respectively, wereapplied to test the potency of antibodies under study. CVS-11 workingstock was amplified and titrated on either BSR or BHK-21, according tothe neutralization test adopted, RFFIT or FAVN, respectively. As well,standard FAVN and RFFIT assays were undertaken to assess the potency oftested antibodies against CVS-11. Briefly, mFAVN assays were based onstandard FAVN but were undertaken on BSR cells.

The cut-off for neutralization was an IC_(go) (pseudotyped viruses) oran IC₅₀ (infectious viruses) above 10000 ng/ml. In other words, if anIC_(go) (pseudotyped viruses) or an IC₅₀ (infectious viruses) above10000 ng/ml was achieved with an antibody, the respective antibody wasconsidered as “not neutralizing”.

Amongst the antigenic site I antibodies tested in the pseudotypedneutralization assay (Wright, E. et al., J Gen. Virol 89, 2204-2213,2008; Wright, E. et al., Vaccine 27, 7178-7186; 2009), RVC20 showed thebest breadth of reactivity being able to neutralize RABV, DUVV, EBLV-1,EBLV-2, ABLV, IRKV, KHUV, ARAV phylogroup I viruses as well as SHIBVfrom phylogroup II and IKOV from putative phylogroup IV (FIG. 5 ). Afull description of the pseudovirus isolates used can be retrieved fromFIG. 1 . As a comparison, the antigenic site I antibody CR57 was clearlyinferior to RVC20, since it was not able to neutralize EBLV-1, SHIBV andIKOV isolates (cf. FIG. 5 ).

When tested on infectious viruses using either the FAVN (Cliquet, F., etal., J. Immunol Methods 212, 79-87, 1998) or the RFFIT (Smith, J. S., etal., Bull. World Health Organ. 48, 535-541, 1973) assays, RVC20 was alsosuperior in its breadth being able to neutralize RABV, DUVV, EBLV-1,EBLV-2, ABLV, BBLV as well as the phylogroup II MOKV (cf. FIG. 6 ; theonly species which was not neutralized is LBV). A full description ofthe infectious virus isolates used can be retrieved from FIG. 1 . In thesame analysis, CR57 did not neutralize EBLV-1 isolates (as observed withpseudoviruses), LBV isolates and MOKV isolates (cf. FIG. 6 ).

Amongst the antigenic site III antibodies tested in the pseudotypedneutralization assay, RVC58 potently neutralized with IC₉₀<10 ng/ml allphylogroup I viruses (i.e. RABV, DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV,ARAV, cf. FIG. 5 ). As a comparison the antigenic site III antibodyCR4098 was far inferior to RVC58, since it was not able to neutralizeDUVV, EBLV-1, EBLV-2, IRKV and KHUV isolates and poorly neutralized ARAV(cf. FIG. 5 ).

When tested on infectious viruses, of all antigenic site III antibodiestested RVC58 was also superior in its breadth, since it was able topotently neutralize RABV, DUVV, EBLV-1, EBLV-2, ABLV, BBLV (cf. FIG. 6 ;the only species which were not neutralized are MOKV and LBV). In thesame analysis CR4098 did not neutralize EBLV-1, DUVV, BBLV, one of thefour RABV isolates tested, one of the three EBLV-2 isolates tested andone of the two ABLV isolates tested (cf. FIG. 6 ).

Of note, antigenic site C antibody RVC68 neutralized all phylogroup Iand II pseudoviruses tested (only WCBV was not neutralized), althoughwith IC₉₀ values 10-100 fold higher as compared to RVC20 and RVC58(FIGS. 5 and 7 ). When tested on infectious viruses, antibody RVC68 was,however, not able to neutralize EBLV, ABLV, MOKV as well as one of thefour RABV isolates tested (FIGS. 6 and 7 ).

If the analysis of the antibody breadth is limited to non-RABVlyssaviruses (scoring as positives all viruses neutralized withIC₅₀<10000 ng/ml), RVC58 (antigenic site III) is able to neutralize 69%of all non-RABV lyssaviruses tested and, remarkably, all the phylogroupI lyssaviruses tested. In comparison antibody CR4098 and RAB1neutralized only 19% and 27%, respectively, of the non-RABV lyssavirusesand 23% and 25%, respectively, of the phylogroup I non-RABVlyssaviruses. In parallel, RVC20 (antigenic site I) is able toneutralize 72% and 91% of the non-RABV lyssaviruses and phylogroup Inon-RABV lyssaviruses, respectively. In comparison antibody CR57neutralized 47% and 68% of the non-RABV lyssaviruses and phylogroup Inon-RABV lyssaviruses, respectively.

When combined, RVC58 and RVC20 covered 78% and 100% of the non-RABVlyssaviruses and phylogroup I non-RABV lyssaviruses, respectively, whileCR57 and CR4098 covered only 50% and 68% of the non-RABV lyssavirusesand phylogroup I non-RABV lyssaviruses, respectively (FIG. 8A-B). HRIGswere also tested against the panel of pseudoviruses and viruses and evenif it is a mixture of polyclonal anti-G protein antibodies covered only25% of the non-RABV lyssaviruses and 36% of the phylogroup I non-RABVlyssaviruses (cf. FIG. 8 ).

To investigate the ability of the antibodies according to the presentinvention to neutralize different RABV isolates in more detail, theanalysis of the neutralizing activity of the antibodies according to thepresent invention RVC20 and RVC58, and of the reference antibodies CR57and CR4098 was then extended to a very large panel of RABV isolates(n=26, 24 viruses and 2 pseudoviruses), which are representative of allcirculating lineages (i.e. American, Asian, Cosmopolitan, Africa 2,Africa 3 and Arctic/Arctic-like lineages) (FIG. 9 ). All 26 RABVisolates were effectively neutralized by RVC20 and RVC58 antibodies withIC₅₀ and IC₉₀ geometric means of 26 and 12 ng/ml, respectively. As acomparison CR57 and CR4098 also neutralized all the RABV tested but withhigher IC₅₀ and IC₉₀ values of 61 and 100 ng/ml, respectively. Of note,CR4098 neutralized two RABV isolates with IC₅₀>10000 ng/ml, aconcentration which is likely not to be effective in vivo.

In a further step, the analysis of the RABV neutralizing activity of theantibodies was further extended, including the further referenceantibody RAB1 and an even larger panel of RABV isolates (n=35, 27viruses and 8 pseudoviruses; CVS-11 was tested as infectious virus andas pseudovirus with FIG. 10 including CVS-11 tested as infectious virusand FIG. 11 showing the results for all three neutralization assaysperformed with CVS-11, namely pseudovirus (PV), FAVN and RFFIT), whichare representative of all circulating lineages (i.e. American, Asian,Cosmopolitan, Africa 2, Africa 3 and Arctic/Arctic-like lineages) (FIG.10B). The full description of the isolates can be retrieved from FIG. 1. As shown in FIG. 10A, all 35 RABV isolates were effectivelyneutralized by RVC20 and RVC58 antibodies with IC₅₀ values (forinfectious viruses) or IC₉₀ values (for pseudoviruses) ranging from 0.1to 140 ng/ml. As a comparison, reference antibodies CR57, CR4098 andRAB1 neutralized all the RABV tested, but with significantly lowerpotency than RVC20 and RVC58 and with a broader range of IC₅₀ or IC₉₀values (i.e. 0.6-969 ng/ml, 0.7-23600 ng/ml, 1-4153 ng/ml, respectively,cf. FIG. 10A). Similarly to RVC20 and RVC58, HRIG neutralized the largemajority of the RABV strains tested with a narrow range of IC₅₀ values.Importantly, CR4098 and RAB1 neutralized six and three RABV isolates,respectively, with an IC₅₀>1000 ng/ml (cf. FIG. 10A), a concentrationwhich is likely not to be effective in post-exposure prophylaxis.

This analysis was extended to additional 8 RABV isolates for which theability of the antibodies to bind to G-protein transfectant cells wastested by flow-cytometry (FIG. 11 ) The full length G genes of RABVstrains were codon optimized for eukaryotic cell expression and clonedinto the phCMV1 vector (Genlantis). G protein expressing plasmids wereused to transfect 293F-Expi cells. Three days after transfection, cellswere collected, fixed and permeabilized with saponin for immunostainingall test antibodies. Binding of antibodies to transfected cells wasanalysed using a Becton Dickinson FACSCanto2 (BD Biosciences) withFlowJo software (TreeStar). As shown in FIG. 11 , all these RABV strainswere recognized by RVC20 and RVC58, whereas RAB1 did not bind to the91001USA strain and CR57 did not bind to RV/R.3PHL/2008/TRa-065 and09029NEP strains. These findings extend the number of RABV isolatesrecognized by RVC20 and RVC58 to 43.

FIG. 10B shows the phylogenetic tree of 2215 RABV G protein sequencesretrieved from public databases. Highlighted with black dots are thesequences of the RABV viruses tested (two G protein sequences, i.e.CV9.13, Mauritania/dog/2019-2006/V6235-2007 were not available and weretherefore not included in the tree). This shows that RABV viruses tested(black dots) are representative of all circulating lineages (i.e.American, Asian, Cosmopolitan, Africa 2, Africa 3 and Arctic/Arctic-likelineages).

A selection of neutralization results using RABV pseudoviruses (PV, thePV neutralization assay was performed according to Wright, E. et al., JGen. Virol 89, 2204-2213, 2008 and Wright, E. et al., Vaccine 27,7178-7186, 2009, which is incorporated by reference herein) orinfectious viruses (as measured by either the fluorescent-antibody virusneutralization test, FAVN, according to Cliquet, F., et al., J. ImmunolMethods 212, 79-87, 1998, which is incorporated by reference herein, orthe rapid fluorescent focus inhibition test, RFFIT, according to Smith,J. S., et al., Bull. World Health Organ. 48, 535-541, 1973, which isincorporated by reference herein) and the characteristics of selectedRABV and non-RABV isolates are shown in FIGS. 12 and 13 , respectively.

Example 5

Epitope Mapping Using Mutant Pseudoviruses.

In order to better refine the epitope specificity of the 12 selectedhuman monoclonal antibodies, they were tested against engineered RABVpseudotypes. In particular, the amino acid changes K226E, K226N, G229E,N336D and N336S found in CR57 and CR4098 viral escape mutants describedin Bakker, A. B. H. et al., J Virol 79, 9062-9068, 2005 and in Marissen,W. et al., J Virol 79, 4672-4678, 2005, were introduced into CVS-11 Ggene and the corresponding mutant pseudoviruses were produced.

The panel of 12 selected antibodies as well as reference antibodies CR57and CR4098 were tested at 15 μg/ml for their ability to neutralize the 5mutant pseudoviruses (K226E, K226N, G229E, N336D and N336S) and comparedwith the corresponding parental CVS-11 strain. The results of thisanalysis are summarized in FIG. 14 . CR57 and RVC20, but not RVC3,antibodies were not able to neutralize the CR57 CVS-11 escape mutantsK226E, K226N and G229E. These results indicate that RVC3 recognizes anepitope in the antigenic site I which is distinct from that recognizedby CR57, and that RVC20 recognize an epitope similar to that recognizedby CR57. However, the finding that RVC20 is characterized by a broaderreactivity against non-RABV lyssaviruses (FIG. 8 ) as compared to CR57indicates that RVC20 antibody mode of recognition of its cognate epitopein the antigenic site I is distinct to that of CR57, being able totolerate a larger number of amino acid changes in the binding site andin the surroundings residues.

All antibodies, including CR4098, with the exception of RAM (data notshown), were able to neutralize the CR4098 CVS-11 escape mutants N336D,thus indicating that this mutation does not have a significant impact onthe binding to their cognate epitopes in the context of the CSV-11 Gprotein. In addition, all the inventive antigenic site III antibodies,RVC58 in particular, showed a greater breadth of reactivity withnon-RABV lyssaviruses as compared to CR4098 (FIG. 7 ).

Example 6

Analysis of the Conservation of RVC20 and RVC58 Epitopes within RABVIsolates.

The antigenic site I recognized by the antibody CR57 was defined bypeptide scanning analysis and by the isolation of viral escape mutantsK226E, K226N, and G229E and found to locate to the minimal bindingregion composed by residues KLCGVL (consensus sequence and positions226-231 of the RABV G protein; Marissen, W. et al., J. Virol 79,4672-4678, 2005). The competition results shown in FIG. 4 and theresults of the mutant pseudovirus test shown in FIG. 14 indicate thatRVC20 binds to the antigenic site I. The present inventors thereforeanalyzed the degree of conservation of the antigenic site I amino acidresidues in a panel of 2566 sequences from independent RABV isolatesretrieved from multiple public databases representative of the globalRABV diversity.

Thereby, it was found that position 226 is a K in 99.73% and R in 0.19%of the sequences analyzed (R or K in 99.92% of the isolates) (FIG. 15A).RVC20, but not CR57, neutralizes viruses carrying both K and R atposition 226 (FIG. 16 ). The other polymorphic position in the antigenicsite I is residue 231, which is L in 67.65%, S in 17.30% and P in 14.73%of the RABV isolates analyzed (L, S or P are present in 99.69% of thesequences, FIG. 15A). RVC20 and CR57 were tested and neutralizedlyssaviruses carrying leucine, serine or proline residues at position231 (FIG. 16 ). This analysis confirmed our previous neutralizationresults and indicated that RVC20 antibody epitope is highly conserved inRABV. Importantly, all three CR57 and RVC20 CVS-11 escape mutants atposition 226 are efficiently neutralized by RVC58.

A similar analysis was performed for the antigenic site III antibodyRVC58. Antigenic site III is primarily formed by residues KSVRTWNEI(consensus sequence and positions 330-338 of the RABV G protein;(Walker, P. J. et al., J. Gen. Virol 80, 1211-1220, 1999; Bakker, A. B.H. et al; J Virol 79, 9062-9068, 2005). The competition results shown inFIG. 4 and the results of the mutant pseudovirus test shown in FIG. 14indicate that RVC58 recognizes residues within the antigenic site III.The present inventors therefore analyzed, as described above for theantigenic site I, the degree of conservation of the antigenic site IIIamino acid residues in a panel of 2566 sequences from independent RABVisolates retrieved from multiple public databases representative of theglobal RABV diversity (as for antigenic site I above).

Thereby, it was found that positions 330, 331, 334, 335 and 337 arehighly conserved (>99.61%), while residues 332, 333, 336 and 338 arepolymorphic (FIG. 15B). Position 330 is a K in 99.61% and N in 0.27% ofthe sequences analyzed (K or N are present in 99.88% of the sequences).RVC58 was shown to neutralize viruses carrying either K or N at position330 (FIG. 16 ). Position 331 is highly conserved being encoded by S in99.96% of the isolates. Position 332 is a V in 77.05% and I in 22.88% ofthe sequences (V or I are present in 99.93% of the isolates). RVC58 wasshown to neutralize lyssaviruses carrying either V or I at position 332.Position 333 is R in 96.22% of the isolates. Several other residues, butnot D, are found at position 333 in RABV isolates. In contrast,phylogroup II lyssaviruses carry a D in that position and these virusesare not neutralized by RVC58, thus suggesting that a D in position 333might compromise RVC58 binding, but this residue is not found in naturalRABV isolates. Position 334 is a T in 99.65% of the isolates. Position335 is W in 100% of the isolates. Position 336 is N in 90.57%, D in3.59%, S in 5.65% and K in 0.08% of the RABV isolates analysed (N, D, Sor K are present in 99.89% of the isolates). RVC58 was shown toneutralize lyssaviruses carrying either N, D, S or K at position 336. Ofnote, RABV carrying D at position 336 are not neutralized by CR4098 andRAB1, thus suggesting that potentially 4% of the circulating RABV areresistant to CR4098 neutralization and to RAB1 neutralization. Of note,the majority of the African RABV isolates analyzed here (59.1%) carry aD at position 336 represent. These isolates correspond to lineageAfrica2. Position 337 is a E in 99.61% and D in 0.35% of the isolates (Eor D are present in 99.96% of the isolates). RVC58 was shown toneutralize lyssaviruses carrying either E or D at position 337. Finally,position 338 is I in 93.73% and V in 6.16% of the isolates analyzed (Ior V are present in 99.9% of the isolates). RVC58 was shown toneutralize lyssaviruses carrying either I or V at position 338.

Thus, RVC58 recognizes RABV and non-RABV isolates carrying multipleresidues in the polymorphic positions that are representative of atleast 99.80% of the RABV analyzed (FIG. 15B, FIG. 16 ). This analysisconfirmed our previous neutralization results wherein RVC58 neutralizedall phylogroup I lyssaviruses tested and indicated that RVC58 antibodyepitope is highly conserved in RABV and non-RABV lyssaviruses.

In summary, the two antibodies RVC58 and RVC20 potently neutralizedhuman and animal RABV isolates as well as most non-RABV lyssaviruses(including the new Eurasian bat viruses) by binding two distinctantigenic sites (site I and III) on the virus G protein. The combinationof these two antibodies represents a treatment with an unprecedentedbreadth of reactivity and with reduced risk of escape mutant selection.

Example 7

RVC58 and RVC20 Antibodies Protect Syrian Hamsters from a Lethal RABVInfection.

To investigate whether the antibodies RVC58 and RVC20 displayneutralizing activity against a lethal RABV infection in vivo, weperformed a Syrian hamster (Mesocricetus auratus) study. At 6 h afteradministration of a lethal dose of RABV CVS-11 (50 μl of 10^(5.7)TCID50/ml in the gastrocnemius muscle of the hind left leg, hamsters(n=12 per group) were left untreated or prophylaxis was initiated witheither vaccine (Imovax; Sanofi-Pasteur: a commercial inactivated humandiploid cell vaccine, which was administered intramuscularly in a volumeof 0.05 ml in the in the gastrocnemius muscle of the hind right leg, adose that correspond to 0.125 international units of rabies antigen)plus HRIG (Berirab®, 20 mg/kg, equivalent to 20 IU/kg and administeredintramuscularly in a volume of 0.05 ml), or vaccine plus 0.045 mg/kg ofan equimolar mixture of RVC20 and RVC58 antibodies or vaccine plus0.0045 mg/kg of an equimolar mixture of RVC20 and RVC58 antibodies.Treated animals also received the rabies vaccine on days, 3, 7, 14 and28. Animals were monitored during the course of the experiment and wereeuthanized when signs of clinical rabies occurred. Eleven out of 12animals that were not treated after infection succumbed by day 8 (FIG.17 ). The standard post-exposure prophylaxis (PEP) based on 20 mg/kgHRIG and vaccine was effective in reducing the overall mortality to 33%(8/12 animals survived; FIG. 17 ). Strikingly, the combination ofRVC58+RVC20 at 0.045 mg/kg (which correspond to 1/440 of theadministered HRIG) protected 75% of the animals (9/12), while a 10 timeslower dose of RVC58 and RVC20 (0.0045 mg/kg) protected only 33% of theanimals. This suggests that 0.045 mg/kg RVC58+RVC20 is superior to the20 mg/kg HRIG dose. The protective dose of 0.045 mg/kg RVC58+RVC20corresponds in humans to an average total dose to be administered duringthe PEP of only 3 mg of the RVC58+RVC20 mixture. This amount could beproduced and formulated in a stable form (i.e. lyophilized formulation,where for instance previous studies have shown that monoclonalantibodies preserved by lyophilization are stable for 33 months at 40°C. and 5 months at 50° C.) and at an affordable cost for developingcountries.

Example 8

RVC58 and RVC20 Antibodies do not Interfere with Vaccination.

During PEP, there is the possibility that the simultaneousadministration of antibodies and vaccine decreases the ability of thevaccine to induce the threshold levels of neutralizing antibodiesrequired for protection. Therefore, it is critical to evaluate thedegree to which an antibody treatment interferes with vaccination. Todetermine the effect of the antibodies mixture on vaccine potency, an invivo animal experiment was performed in the absence of RABV challenge.In particular, all animals (n=12 per group) were vaccinated with rabiesvaccine on day 0, 3, 7, 14 and 28 (Imovax, Sanofi-Pasteur, administeredintramuscularly in a volume of 0.05 ml in the in the gastrocnemiusmuscle of the hind right leg, a dose that correspond to 0.125international units of rabies antigen) and concomitantly administered onday 0 with HRIG (Berirab®, 20 mg/kg) or an equimolar mixture ofRVC58+RVC20 at 0.045 mg/kg or 40 mg/kg (888 times higher dose) that wereinjected intramuscularly in the in the gastrocnemius muscle of the hindleft leg. Serum binding titers (measured in ELISA on RABV G-proteincoated plates by detecting the G-protein-bound hamster antibodies withalkaline-phosphatase-conjugated anti-hamster polyclonal antibodies),serum neutralizing titers (neutralization FAVN assay on CVS-11;according to Cliquet, F., et al., J. Immunol Methods 212, 79-87, 1998)and levels of residual human IgG antibodies were determined on day 42.HRIG and 0.045 mg/kg of RVC58+RVC20 did not reduce the endogenoushamster IgG binding antibody response to the RABV G protein (FIG. 18A)as compared to animals receiving vaccine alone. Of note, the level ofneutralizing antibodies in animals treated with both the 0.045 and the40 mg/kg doses is comparable to that elicited by the vaccine alone or bythe vaccine and HRIG treated animals and in most animals theneutralizing titer is above 10 IU/ml (FIG. 18B). Finally, while stillhigh levels of human antibodies (above 10 μg/ml) are found on day 42 inanimals treated with 20 mg/kg of HRIG or 40 mg/kg of RVC58+RVC20,undetectable to low levels of human IgG were found in the sera ofanimals treated with 0.045 mg/kg of RVC58+RVC20 (FIG. 18C). Theseresults suggest that a dose of 0.045 mg/kg RVC58+RVC20, which was shownto be protective, does not compromise the production of virusneutralizing antibodies elicited in animals upon RABV vaccination.

Example 9

RVC58 and RVC20 Antibodies Act Therapeutically in Syrian HamstersLethally Infected with RABV.

Currently, there is no treatment for rabies. The development of atreatment would be of benefit for at least two classes of patients:those with known exposure to RABV but who have failed to receive promptpost-exposure prophylaxis due to circumstances and who are at increasedrisk of developing RABV infection, and those who did not recognizecontact with the virus and present signs (of different severity) of thedisease (e.g. individuals infected by unnoticed contacts with infectedbats; RABV of bat origin where dog rabies is controlled has become theleading cause of human rabies). Single or multiple i.v. injections withthe RVC58 and RVC20 cocktail (i.e. an equimolar mixture of RVC58 andRVC20 antibodies) would provide high titres of systemic neutralisingantibodies (including in the CNS) and block viral replication anddisease progression. The development of a cocktail of potent and broadlyneutralizing antibodies may help to expand the post-exposure treatmentwindow for human RABV infection, that is currently limited to the firstdays after infection. In these individuals the RV might has alreadyreached the CNS tissues and early or late signs of the disease mighthave also appeared. These patients could benefit from a treatment withhighly potent neutralizing antibodies that can leak across the bloodbrain barrier (or administered directly in the CSN) delivering asufficient amount of antibodies capable of effectively neutralizing thevirus replication in the CNS tissue.

The therapeutic potential of RVC58+RVC20 antibodies was evaluated inSyrian hamsters lethally challenged with a field RABV isolate. Inparticular, RVC58+RVC20 were tested in Syrian hamsters challenged in thegastrocnemius muscle of a back leg with a lethal dose of a field virusisolated from the salivary glands of an infected fox (Italy/redfox/673/2011). In infected animals, RABV was detectable in the CNS(central nervous system) on day 5 after challenge. Animals were treatedwith a single injection of 40 mg/kg of RVC58+RVC20 given either on day 1(n=12), on day 5 (n=12) or on day 9 (n=7) after infection without aconcomitant administration of the vaccine. Control groups receivedeither phosphate-buffered saline (n=17) or the standard PEP (20 mg/kgHRIG and vaccine; n=12). Animals were monitored twice daily andeuthanized when clinical signs of rabies appeared. Strikingly,RVC58+RVC20 protected animals from lethal infection when administered upto 5 days after infection (FIG. 19 ). Of note, 3 out of the 12 treatedanimals showed clinical signs of the disease (paralysis of back leg siteof challenge), which however did not develop further. In this model theclassical PEP conferred only a modest protection as compared tountreated animals (FIG. 19 ). No signs of disease were detected insurviving animals up to 60 days after infection.

In all succumbed animals and in all survivors (which were sacrificed onday 60) the presence of RABV was revealed by quantifying the genomic RNAand viral mRNA encoding for the N protein in spinal cord, medullaoblongata/cerebellum and brain quantified using quantitative real-timePCR. Of note, detectable levels of viral RNA were measured in the CNS ofasymptomatic animals treated with RVC58+RVC20 on day 1 or 5 afterinfection (albeit at levels 100-1000 lower than those measured insuccumbing animals) (FIG. 20A), thus indicating that the initial RABVinfection was not abortive but kept under control within the CNS by theadministered highly potent neutralizing antibodies and most likely by aconcomitant endogenous immune response to the virus.

The development of a robust endogenous immune response was alsoconfirmed by the measurement of RABV G-protein-specific hamster IgGantibody titers in the sera of all animals (FIG. 20B). Of note, animalsreceiving RVC58+RVC20 on day 5 (all survived the lethal infection)developed high levels of G-protein-specific IgG antibodies at levelscomparable, or higher, than those elicited in surviving animals by thevaccine in the PEP group. The level of these antibodies was alsocomparable or higher than those elicited in unchallenged animalsreceiving the standard PEP (see FIG. 18 ). Finally, the high dose ofRVC58+RVC20 might also be compatible with the concomitant vaccination asshown by the finding that the use of a high dose of these antibodies donot compromise the immune response to the vaccine (FIG. 18A-B).

Tissue samples from the brain, medulla oblongata and spinal cord ofsymptomatic control animals or animals receiving RVC58+RVC20 on day 5(and sacrificed on day 60) were analyzed for the presence of RABV Nantigen by immunohistochemistry (IHC). In particular, the IHC analysiswas focused on the identification of Negri bodies, which areeosinophilic, sharply outlined, pathognomonic inclusion bodies (2-10 μmin diameter) made by aggregates of nucleocapsids and found in thecytoplasm of neurons containing the rabies virus. While numerous Negribodies were found in CNS tissues from positive control animals, onlyvery few bodies were identified in animals treated with antibodies onday 5 (FIG. 21 ). These results confirm that RABV has reached the CNS,and even the brain, in animals treated with the high dose of RVC58+RVC20without causing symptoms.

The presence of RABV neutralizing antibodies early in patients clinicalcourse is considered an important factor contributing to a favorableoutcome. This probably occurs in less than 20% of all patients withrabies. The presence of RABV neutralizing antibodies is a marker of anactive adaptive immune response that is essential for viral clearance(Lafon, M., in “Rabies”, A. C. Jackson and W. H. Wunner, 3^(rd) eds.,pp. 489-504, Elsevier Academic Press, London, 2013). There have been sixsurvivors of rabies who received rabies vaccine prior to the onset oftheir disease (and only one who did not receive vaccine). This supportsthe notion that an early immune response is associated with a positiveoutcome. Finally, most survivors of rabies have shown RABV neutralizingantibodies in sera and cerebrospinal fluid. The potent and broad humanRABV neutralizing antibodies according to the present invention, forexample RVC20 and RVC58, offer the opportunity to confer an immediateand robust passive immunity, which might represent (i) a potent agentfor post-exposure therapy, which is effective at much lowerconcentrations compared to HRIG and (ii) a valid therapeutic agent forthe treatment of patients with an early clinical diagnosis of rabies. Inthis regard it is conceivable that a prompt initiation of therapy mightoffer the best opportunity for a favorable outcome. The antibodiesaccording to the present invention, for example the human monoclonalantibodies RVC58 and RVC20, can therefore represent an effective therapyalone or in combination with other therapies including rabiesvaccination, ribavirin (or other antivirals), interferon-alpha andketamine.

Table of Sequences and SEQ ID Numbers SEQ ID NO Description Sequence*RVA122 ANTIBODY   1 CDRH1 aa G D S MNNF Y   2 CDRH2 aa IYYSG T T   3CDRH3 aa ARD S GDYVSYYYYGMDV   4 CDRL1 aa SSNIGSNY   5 CDRL2 aa KSD   6CDRL2 long aa LIY KSD KRP   7 CDRL3 aa AAWD NR LSGW L   8 CDRH1 nucGGTGACTCCATGAATAATTTCTAC   9 CDRH2 nuc ATCTATTACAGTGGGACCACC  10CDRH3 nuc GCGAGAGACTCCGGTGACTACGTCAGCTACTACTATTATG GTATGGACGTC  11CDRL1 nuc AGCTCCAACATCGGAAGTAATTAT  12 CDRL2 nuc AAGAGTGAT  13CDRL2 long nuc cttatttacAAGAGTGATaagcggccc  14 CDRL3 nucGCAGCATGGGATAACAGGCTGAGTGGTTGGCTC  15 heavy chain aaQVHLQESGPGLVKPSETLSLTCTVSG D S MNNF YWGWIRQP AGKGLEWIGYIYYSG TTNYNPSLKSRVTISIDTSKNQFSL KVNSVTAADTAVYYCARD S GDYVSYYYYGMDVWGPGTTVT VSS 16 light chain aa QSVLTQSPSASDTPGQRVTISCSGSSSNIGSNYVYWYQQF PGTAPKLLIYKSDKRPSGVPDRFSGSTSGTSASLAISGLR SEDEADYYCAAWD NR LSGW L FGGGTKLTVL  17heavy chain nuc caggtgcacctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctGGTGACTCCATGAATAATTTCTACtggggctggatccggcagcccgcagggaagggactggagtggattggatatATCTATTACAGTGGGACCACCaactacaacccctccctcaagagtcgagtcaccatatcaatagacacgtccaagaaccaattctccctgaaggtgaactctgtgaccgctgcggacacggccgtgtattattgtGCGAGAGACTCCGGTGACTACGTCAGCTACTACTATTATGGTATGGACGTCtggggcccagggaccacggtcacc gtctcctcag  18 light chain nuccagtctgtgctgactcagtcaccctcagcgtctgatacccccgggcagagggtcaccatctcttgttctggaagcAGCTCCAACATCGGAAGTAATTATgtgtattggtaccagcagttcccaggaacggcccccaaactccttatttacAAGAGTGATaagcggccctcaggggtccctgaccgattctctggctccacgtctggcacctcagcctccctggccatcagtgggctccggtccgaagatgaggctgattattactgtGCAGCATGGGATAACAGGCTGAGTGGTTGGCTCttcggcggagggacgaagct gaccgtcctag RVA144 ANTIBODY  19CDRH1 aa GGSISS TIF Y  20 CDRH2 aa V YY N G H T  21 CDRH3 aaARPSTYDYSIGR  22 CDRL1 aa SSNIGAGYD  23 CDRL2 aa GN T  24 CDRL2 long aaLIYGN TKRP  25 CDRL3 aa QS F DSSLS A WV  26 CDRH1 nucGGTGGTTCCATCAGCAGTACTATTTTCTAC  27 CDRH2 nuc GTCTATTATAATGGACACACC  28CDRH3 nuc GCGAGACCCTCAACATATGACTACAGTATTGGGCGC  29 CDRL1 nucAGCTCCAACATCGGGGCAGGTTATGAT  30 CDRL2 nuc GGTAACACC  31 CDRL2 long nucctcatctatGGTAACACCaagcggccc  32 CDRL3 nucCAGTCCTTTGACAGCAGCCTGAGTGCTTGGGTA  33 heavy chain aaQLQLQESGPGLVKPSETLSLTCTVSGGSISS TIF YWGWIR QPPGKGLEWIGS V YY N G HTYYNPSLKSRVAISIDKSKNQF SLRLNSVTAADTAVYYCARPSTYDYSIGRWGQGTLVTVSS  34light chain aa QSVLTQPPSVSRAPGQRVTISCTGSSSNIGAGYDVHWYQQ LPGTAPKLLIYGNTKRPSGVPDRFSGSKSGTSASLAITGL LTEDEADYYCQS F DSSLS A WVFGGGTKLTVL  35heavy chain nuc cagctgcagctgcaggagtcgggcccaggactggtgaagccctcggagaccctgtccctcacttgcactgtctctGGTGGTTCCATCAGCAGTACTATTTTCTACtggggctggatccgccagcccccagggaagggactggagtggattgggagtGTCTATTATAATGGACACACCtactacaatccgtccctcaagagtcgagtcgccatatccattgacaagtccaagaaccagttctccctgaggcttaactctgtgaccgccgcggacacggctgtatattactgtGCGAGACCCTCAACATATGACTACAGTATTGGGCGCtggggccagggaaccctggtcaccgtctcctca g  36 light chain nuccagtccgtgctgacgcagccgccctcagtgtctcgggccccagggcagagggtcaccatctcctgcactgggagcAGCTCCAACATCGGGGCAGGTTATGATgtccactggtaccagcaacttccaggaacagcccccaaactcctcatctatGGTAACACCaagcggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcactgggctcctgactgaggatgaggctgattattactgccAGTCCTTTGACAGCAGCCTGAGTGCTTGGGTAttcggcggagggaccaa actgaccgtcctgg RVB185 ANTIBODY 37 CDRH1 aa G APV S GVNS Y  38 CDRH2 aa I K YSGST  39 CDRH3 aa ARQS TMTGRDY  40 CDRL1 aa R SNIGS HP  41 CDRL2 aa GDS  42 CDRL2 long aa LIY GDSQRP  43 CDRL3 aa AAWDDSL S GLWV  44 CDRHi nucGGTGCCCCCGTCAGTGGTGTTAACTCCTAC  45 CDRH2 nuc ATCAAGTACAGTGGGAGCACC  46CDRH3 nuc GCCAGACAAAGTACTATGACGGGCCGGGACTAC  47 CDRL1 nucAGATCCAACATCGGAAGCCATCCT  48 CDRL2 nuc GGTGATAGT  49 CDRL2 long nucctcatctatGGTGATAGTcagcgaccc  50 CDRL3 nucGCAGCATGGGATGACAGCCTGAGTGGCCTTTGGGTG  51 heavy chain aaQVQLQESGPGLVKPSETLSLTCSVSG APV S GVNS YWVWIR QPPGKGLEWIAT I K YSGSTHRSPSLRSRVTISVDTSKNQF SLELSSVTAADTAVYYCARQS TM TGRDYWGQGTLVTVSS  52light chain aa QSVLTQPPSASGTPGQRVTISCSGS R SNIGS HP VNWYQQL PGAAPKLLIYGDS QRPSGVPDRFSGSKSGPSASLAISGLQ SEDEADYYCAAWDDSL S GLWVFGGGTKLTVL  53heavy chain nuc caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcagtgtctccGGTGCCCCCGTCAGTGGTGTTAACTCCTACtgggtgtggatccgccagccccccgggaaggggctggagtggattgcgactATCAAGTACAGTGGGAGCACCcaccgtagcccgtcgctcaggagtcgagtcaccatatccgtagacacgtccaagaatcagttctccctggagctgagctctgtgaccgccgctgacacggctgtatattactgtGCCAGACAAAGTACTATGACGGGCCGGGACTACtggggccagggaaccctggtcaccgtctcctcag  54 light chain nuccagtctgtgctgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgttctggaagcAGATCCAACATCGGAAGCCATCCTgtaaactggtaccagcagctcccgggagcggcccccaagctcctcatctatGGTGATAGTcagcgaccctcaggggtccctgaccgattctctggctccaagtctggcccctcagcctccctggccatcagtggactccagtctgaagatgaggctgattattactgtGCAGCATGGGATGACAGCCTGAGTGGCCTTTGGGTGttcggcggagggaccaa gctgaccgtcctaa RVB492 ANTIBODY 55 CDRH1 aa GF S FSSYA  56 CDRH2 aa LNSIDHR T  57 CDRH3 aaARGVGLWFGELSWNYFDY  58 CDRL1 aa S N D I GGYNY  59 CDRL2 aa Y V N  60CDRL2 long aa MIFY V N KRP  61 CDRL3 aa CS F AGSY S L  62 CDRH1 nucGGATTCAGCTTTAGCAGCTATGCC  63 CDRH2 nuc CTTAATTCTATTGATCATAGAACA  64CDRH3 nuc GCTCGGGGGGTGGGACTATGGTTCGGTGAATTATCCTGGA ATTACTTTGACTAC  65CDRL1 nuc AGCAATGATATTGGTGGTTATAACTAT  66 CDRL2 nuc TATGTCAAT  67CDRL2 long nuc atgatttttTATGTCAATaagcggccc  68 CDRL3 nucTGCTCATTTGCAGGCAGTTACTCCTTA  69 heavy chain EVQLMESGGGLVQPGGSMRLYCAASGFS FSSYAMSWVRQA variant 1 aa PGKGLEWVSSLNSIDHR T DYADSVKGRFTISRDNSKNTLYLQMDSLRVEDSAMYYCARGVGLWFGELSWNYFDYWGQGTL VTVSS  70 heavy chainEVQLVQSGGGLVQPGGSMRLYCAASGF S FSSYAMSWVRQA variant 2 aaPGKGLEWVSSLNSIDHR T DYADSVKGRFTISRDNSKNTLYLQMDSLRVEDSAMYYCARGVGLWFGELSWNYFDYWGQGTL VTVSS  71 light chain aaQSALTQPRSVSGSPGQSVTISCTGTS N D I GGYNYVSWYQQ HPGKAPKLMIFY V NKRPSGVPDRFSGSKSGNTASLTISGL QAEDEADYYCCS F AGSY S LFGRGTKLTVL  72heavy chain gaggtgcagctgatggagtctgggggaggcctggtacagc variant 1 nuccgggggggtccatgagactctactgtgcagcctctGGATTCAGCTTTAGCAGCTATGCCatgagctgggtccgccaggctccagggaaggggctcgagtgggtctcaagtCTTAATTCTATTGATCATAGAACAgactatgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacaccctgtatttacaaatggacagcctgagagtcgaggactcggccatgtattactgtGCTCGGGGGGTGGGACTATGGTTCGGTGAATTATCCTGGAATTACTTTGACTACtggggccagggaaccctg gtcaccgtctcctcag  73heavy chain gaggtgcagctggtgcagtctgggggaggcctggtacagc variant 2 nuccgggggggtccatgagactctactgtgcagcctctGGATTCAGCTTTAGCAGCTATGCCatgagctgggtccgccaggctccagggaaggggctcgagtgggtctcaagtCTTAATTCTATTGATCATAGAACAgactatgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacaccctgtatttacaaatggacagcctgagagtcgaggactcggccatgtattactgtGCTCGGGGGGTGGGACTATGGTTCGGTGAATTATCCTGGAATTACTTTGACTACtggggccagggaaccctg gtcaccgtctcctcag  74light chain nuc cagtctgccctgactcagcctcgctcagtgtccgggtctcctggacagtcagtcaccatctcctgcactggaaccAGCAATGATATTGGTGGTTATAACTATgtctcctggtaccaacaacacccaggcaaagcccccaaactcatgatttttTATGTCAATaagcggccctcaggggtccctgatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggatgaagctgattattactgcTGCTCATTTGCAGGCAGTTACTCCTTAttcggcagagggaccaagctgac cgtcctag RVC3 ANTIBODY  75CDRH1 aa T FTF RN YA  76 CDRH2 aa IS A SG S ST  77 CDRH3 aaAKFAHDFWSGYSYFD S  78 CDRL1 aa QSV N SN  79 CDRL2 aa GAS  80CDRL2 long aa LIYGASTRA  81 CDRL3 aa QQYNNWVSIT  82 CDRH1 nucACATTCACGTTTAGAAACTATGCC  83 CDRH2 nuc ATTAGTGCTAGTGGTAGTAGCACG  84CDRH3 nuc GCGAAATTTGCTCACGATTTTTGGAGTGGTTATTCTTACT TTGACTCC  85CDRL1 nuc CAGAGTGTTAACAGCAAC  86 CDRL2 nuc GGTGCATCC  87 CDRL2 long nucctcatctatGGTGCATCCaccagggcc  88 CDRL3 nuc CAGCAGTATAATAATTGGGTTTCGATCACC 89 heavy chain aa EVQLLESGGGLVQPGGSLRLSCAAS T FTF RN YAMSWVRQAPGKGLDWVSG IS A SG S ST NYAASLKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFAHDFWSGYSYFD S WGQGTLVT VSS  90 light chain aaEIVMTQSPATLSVSPGERATLSCRAG QSV N SNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQS EDFAVYYCQQYNNWVSITFGQGTRLEIK 91 heavy chain nuc gaggtgcagctgttggagtctgggggaggcctggtgcagccgggggggtccctgagactctcctgtgcagcctctACATTCACGTTTAGAAACTATGCCatgtcctgggtccgccaggctccagggaaggggctggactgggtctcagggATTAGTGCTAGTGGTAGTAGCACGaattatgcagcctccctgaagggccgatttaccatctccagagacaattccaagaacacattgtatctgcaaatgaacagcctgagagccgaggacacggccgtctattactgtGCGAAATTTGCTCACGATTTTTGGAGTGGTTATTCTTACTTTGACTCCtggggccagggaaccctggtcacc gtctcctcag  92 light chain nucgaaatagtgatgacgcagtctccagccaccctgtctgtgtctccaggggaaagagccaccctctcctgcagggccggtCAGAGTGTTAACAGCAACttagcctggtaccagcagaaacctgggcaggctcccagactcctcatctatGGTGCATCCaccagggccactggtatcccagccaggttcagtggcagtgggtctgggacagagttcactctcaccatcagcagcctgcagtctgaagattttgcagtttattactgtCAGCAGTATAATAATTGGGTTTCGATCACCttcggccaagggacacgactggagat taaac RVC20 ANTIBODY  93CDRH1 aa GGS F SSGSY S  94 CDRH2 aa IYYSGST  95 CDRH3 aa ARGTY S DFWSGSPL DY  96 CDRL1 aa QGISNY  97 CDRL2 aa AAS  98 CDRL2 long aa LIYAASSLQ 99 CDRL3 aa QQY DT YPLT 100 CDRH1 nuc GGTGGCTCCTTCAGCAGTGGAAGTTACTCC101 CDRH2 nuc ATCTATTACAGTGGGAGCACT 102 CDRH3 nucGCGAGAGGCACGTATTCCGATTTTTGGAGTGGTTCCCCTT TAGACTAC 103 CDRL1 nucCAGGGCATTAGCAATTAT 104 CDRL2 nuc GCTGCATCC 105 CDRL2 long nucctgatctatGCTGCATCCagtttgcaa 106 CDRL3 nuc CAACAGTATGATACTTACCCTCTCACT107 heavy chain aa QVQLQESGPGLVKPSQTLSLTCTVSGGS F SSGSY S WNWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSRVTMSVHTSKNQF SLKLNSITAADTAVYYCARGTY SDFWSGSP L DYWGQGTLV TVSS 108 light chain aaDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTINSLQP EDFVTYFCQQY DT YPLTFGGGTKVEIK109 heavy chain nuc caggtgcagctgcaggagtcgggcccaggactggtgaagccttcacagaccctgtccctcacctgcactgtctccGGTGGCTCCTTCAGCAGTGGAAGTTACTCCtggaactggatccgccagcacccagggaagggcctggagtggattgggtacATCTATTACAGTGGGAGCACTtattacaacccgtccctcaagagtcgagttaccatgtcagtacacacgtctaagaaccagttctccctgaagctgaactctataactgccgcggacacggccgtgtattactgtGCGAGAGGCACGTATTCCGATTTTTGGAGTGGTTCCCCTTTAGACTACtggggccagggaaccctggtc accgtctcctcag 110light chain nuc gacatccagatgacccagtctccatcctcactgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtCAGGGCATTAGCAATTATttagcctggtttcagcagaaaccagggaaagcccctaagtccctgatctatGCTGCATCCagtttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcaccatcaacagcctgcagcctgaagattttgtaacttatttctgcCAACAGTATGATACTTACCCTCTCACTttcggcggagggaccaaggtggagatcaa ac RVC21 ANTIBODY 111 CDRH1 aaGGSIS NPN YY 112 CDRH2 aa IYY N G Y T 113 CDRH3 aa ATQST M TTIAGH Y 114CDRL1 aa T SNIGN S Y 115 CDRL2 aa DNN 116 CDRL2 long aa LIYDNNKRP 117CDRL3 aa GTWDSSL N AYV 118 CDRH1 nuc GGTGGCTCCATCAGCAACCCTAACTACTAC 119CDRH2 nuc ATCTATTATAATGGGTACACC 120 CDRH3 nucGCGACGCAATCTACGATGACTACCATAGCGGGCCACTAC 121 CDRL1 nucACATCCAACATTGGGAATTCTTAT 122 CDRL2 nuc GACAATAAT 123 CDRL2 long nucctcatttatGACAATAATaagcgaccc 124 CDRL3 nucGGAACATGGGACAGCAGCCTGAATGCTTATGTC 125 heavy chain aaQLQLQESGPGLVKPSETLSLTCTVSGGSIS NPN YYWGWIR QPPGKGLEWIGSIYY N G YTYYNPSLKSRVTISVDKSKDQF FLKMTSLTAADTAVYYCATQST M TTIAGH Y WGQGTLVTVS S126 light chain aa QSVLTQAPSVSAAPGLKVTISCSGS T SNIGN S YVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSDTSATLGITGLQ TGDEADYYCGTWDSSL NAYVFGTGTKVTVL 127 heavy chain nuccagctgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacgtgcactgtctctGGTGGCTCCATCAGCAACCCTAACTACTACtggggctggatccgccagcccccagggaagggtctggaatggattgggagtATCTATTATAATGGGTACACCtactacaacccgtccctcaagagtcgagttaccatatccgtggacaagtccaaggaccagttctttctgaagatgacctctctgaccgccgcagacacggctgtgtattactgtGCGACGCAATCTACGATGACTACCATAGCGGGCCACTACtggggccagggaaccctggtcaccgtctcc tcag 128 light chain nuccagtctgtattgacgcaggcgccctcagtgtctgcggccccaggactaaaggtcaccatctcctgctctggaagcACATCCAACATTGGGAATTCTTATgtatcctggtaccagcagctcccaggaacagcccccaaactcctcatttatGACAATAATaagcgaccctcagggattcctgaccgattctctggctccaagtctgacacgtcagccaccctgggcatcaccggactccagactggggacgaggccgattattactgcGGAACATGGGACAGCAGCCTGAATGCTTATGTCttcggaactgggaccaaggt caccgtcctag RVC38 ANTIBODY 129CDRH1 aa GGTFSSYA 130 CDRH2 aa I M P M F VA A 131 CDRH3 aaARGDGYNYKWYFDL 132 CDRL1 aa Q D IS N Y 133 CDRL2 aa AAS 134CDRL2 long aa LIYAASTLQ 135 CDRL3 aa QQL DT YVALT 136 CDRH1 nucggaggcaccttcagcagctatgcc 137 CDRH2 nuc atcatgcctatgtttgtggcggca 138CDRH3 nuc gcgagaggggatggctacaattacaagtggtattttgacc tt 139 CDRL1 nuccaggacattagtaattat 140 CDRL2 nuc gctgcatcc 141 CDRL2 long nucctgatctatgctgcatccactttgcaa 142 CDRL3 nuc caacagcttgatacttacgtcgcgctcact143 heavy chain aa EVQLVQSGAEVKKPGSSVRVSCKASGGTFSSYAISWVRQA PGLGLEWMGGIM P M F VA ANYAQNFQGRVTVSVDKSTNTAYMEMHNLRSDDTAMYYCARGDGYNYKWYFDLWGQGTLVTVS S 144 light chain aaDIQLTQSPSFLSASVGDRVTITCRASQ D IS N YLAWYQQKPGKPPKLLIYAASTLQRGVPSRFSGSGSGSEFTLTISSLQP EDTATYYCQQL DT YVALTFGGGTKVEIK145 heavy chain nuc gaggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgagggtctcctgcaaggcttctggaggcaccttcagcagctatgccatcagctgggtgcgacaggcccctgggctagggcttgagtggatgggagggatcatgcctatgtttgtggcggcaaactacgcacagaacttccagggcagagtcacggtttctgtggacaaatccacgaacaccgcctatatggagatgcacaacctgagatctgacgacacggccatgtattactgtgcgagaggggatggctacaattacaagtggtattttgacctttggggccagggaaccctagtcaccgtctcc tcag 146 light chain nucgacatccagttgacccagtctccatccttcctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcaggacattagtaattatttagcctggtatcagcaaaaaccagggaagccccctaaactcctgatctatgctgcatccactttgcaaaggggggtcccatcaaggttcagtggcagtggatctgggtcagaattcactctcacaatcagcagcctgcagcctgaagattttgcaacttattactgtcaacagcttgatacttacgtcgcgctcactttcggcggagggaccaaggtggagat caaac RVC44 ANTIBODY 147CDRH1 aa GFTFSSYS 148 CDRH2 aa IS TTGT YI 149 CDRH3 aa ARRSAIA LAGTQRAFDI 150 CDRL1 aa Q N I NN Y 151 CDRL2 aa AAS 152 CDRL2 long aaLIYAASSLH 153 CDRL3 aa QQSYS N PWT 154 CDRH1 nucGGCTTCACCTTTAGTAGTTATAGT 155 CDRH2 nuc ATCAGTACTACTGGTACTTACATA 156CDRH3 nuc GCGAGACGGTCGGCCATAGCACTGGCTGGTACGCAGCGTG CTTTTGATATC 157CDRL1 nuc CAGAACATTAACAACTAT 158 CDRL2 nuc GCTGCATCC 159 CDRL2 long nucctgatctatGCTGCATCCagtttacat 160 CDRL3 nuc caacagagttacagtaacccttggacg161 heavy chain aa EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYSMSWVRQA PGKGLEWVSSISTTGT YIYYADSVEGRFSISRDSARSSLF LQMNSLRAEDTAVYYCARRSAIA L AGTQRAFDIWGPGTNVIVSS 162 light chain aa DIQMTQSPSSLSASVGDRVTITCRASQ N I NN YLNWYQQKLGKAPKLLIYAASSLHSGVPSRFSASGSGTDFILTISNLQP EDCATYYCQQSYS N PWTFGQGTKVEIK163 heavy chain nuc gaggtgcagctggtgcagtctgggggaggcctggtcaagcctggggggtccctgagactctcctgtgcagcctctGGCTTCACCTTTAGTAGTTATAGTatgagttgggtccgccaggctccagggaagggcctggagtgggtctcatccATCAGTACTACTGGTACTTACATAtactacgcagactcagtggagggccgattctccatttccagagacagcgccaggagctctctgtttctgcaaatgaacagcctgagagccgaggacacggctgtctattactgtGCGAGACGGTCGGCCATAGCACTGGCTGGTACGCAGCGTGCTTTTGATATCtggggcccagggacaaacgtc atcgtctcttcag 164light chain nuc gacatccagatgacccagtctccatcttccctgtctgcatctgtaggagacagagtcaccatcacttgccgggcaagtCAGAACATTAACAACTATttaaattggtatcagcagaaactagggaaagcccctaagctcctgatctatGCTGCATCCagtttacatagtggggtcccatcaaggttcagtgccagtggatctgggacagatttcattctgaccatcagtaatctgcaacctgaagattgtgcaacttactactgtcaacagagttacagtaacccttggacgttcggccaagggaccaaggtggaaatcaa ac RVC58 ANTIBODY 165 CDRH1 aaGFTFS T YA 166 CDRH2 aa IS DR GGS R 167 CDRH3 aa A R DIA P PY N YY FYGMDV 168 CDRL1 aa SSD I G AF NY 169 CDRL2 aa EVS 170 CDRL2 long aaIIYEVSNRP 171 CDRL3 aa N SYTSSSTQL 172 CDRH1 nucGGATTCACCTTTAGCACCTATGCC 173 CDRH2 nuc ATTAGTGATAGAGGTGGTAGTAGA 174CDRH3 nuc GCGAGAGATATTGCCCCCCCATATAACTACTACTTCTACG GTATGGACGTC 175CDRL1 nuc AGCAGTGACATTGGTGCTTTTAACTAT 176 CDRL2 nuc GAGGTCAGT 177CDRL2 long nuc ataatttatGAGGTCAGTaatcggccc 178 CDRL3 nucAACTCATATACAAGCAGCAGCACTCAGTTA 179 heavy chain aaEVQLVESGGGLVQPGGSLRLSCAASGFTFS T YAMNWVRQA PGKGLEWVSG IS DR GGS RYYAGSVKGRFTISRDNSKNTLF LQMNSLRAEDTAVYYCA R DIA P PY N YY F YGMDVWGRGTTVTVSS 180 light chain aa QSALTQPASVSGSPGQSITISCTGTSSD I G AF NYVSWYQQHPGKAPKLIIYEVSNRPSGVSNRFSGSKSGNTASLTISGL QAEDEADYYC NSYTSSSTQLFGGGTKLTVL 181 heavy chain nucgaggtgcagctggtggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcggcctctGGATTCACCTTTAGCACCTATGCCatgaattgggtccgccaggctccagggaaggggctggagtgggtctcaggtATTAGTGATAGAGGTGGTAGTAGAtactacgcaggctccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtttctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtGCGAGAGATATTGCCCCCCCATATAACTACTACTTCTACGGTATGGACGTCtggggccgagggaccacggtc accgtctcctcag 182light chain nuc cagtctgccctgactcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggtaccAGCAGTGACATTGGTGCTTTTAACTATgtctcttggtaccaacagcacccaggcaaagcccccaaactcataatttatGAGGTCAGTaatcggccctcaggggtttctaatcgcttctctggctccaagtctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattattactgcAACTCATATACAAGCAGCAGCACTCAGTTAttcggcggagggaccaagct gaccgtcctag RVC68 ANTIBODY 183CDRH1 aa GGSIS EHH 184 CDRH2 aa I FH SGST 185 CDRH3 aa ARAV S TYYYYY IDV 186 CDRL1 aa Q D IS N W 187 CDRL2 aa AAS 188 CDRL2 long aa LIYAASSLQ189 CDRL3 aa QQA K SFPLT 190 CDRH1 nuc GGTGGCTCCATTAGTGAGCACCAC 191CDRH2 nuc ATCTTTCACAGTGGGAGTACC 192 CDRH3 nucGCGAGAGCGGTGTCTACTTACTACTACTATTACATAGACG TC 193 CDRL1 nucCAGGATATTAGCAACTGG 194 CDRL2 nuc GCTGCGTCC 195 CDRL2 long nucctgatctatGCTGCGTCCagtttgcaa 196 CDRL3 nuc CAACAGGCTAAGAGTTTCCCTCTTACT197 heavy chain aa QVQLQESGPRLVKPSETLSLTCTFSGGSIS EHH WSWLRQSPGKGLEWIGYI FH SGSTNYNPSLKSRVNISLDKSKNQFSE KLSSVTAADTAVYFCARAV S TYYYYYI DVWGQGTTVTVSS 198 light chain aa DIQMTQSPSSVSASVGDRVTITCRASQ D IS NWLAWYQQKP GKAPKLLIYAASSLQSGISSRFSGGGSGTDTTLTISSLQP EDFASYYCQQA KSFPLTFGQGTKLEIK 199 heavy chain nuccaggtgcagctacaggagtcgggcccaagactggtgaagccctcggagaccctgtccctcacctgcactttctctGGTGGCTCCATTAGTGAGCACCACtggagctggctccggcagtccccagggaagggactggagtggattggatatATCTTTCACAGTGGGAGTACCaactacaacccctccctcaagagtcgagtcaacatatcattagacaagtccaagaaccagttctccctgaagctgagttctgtgaccgctgcggacacggccgtgtatttctgtGCGAGAGCGGTGTCTACTTACTACTACTATTACATAGACGTCtggggccaagggaccacggtcaccgtctcctca g 200 light chain nucgacatccagatgacccagtctccatcttccgtgtctgcatctgtaggagacagagtcaccatcacttgtcgggcgagtCAGGATATTAGCAACTGGttagcctggtatcagcagaaaccagggaaagcccctaaactcctgatctatGCTGCGTCCagtttgcaaagtgggatctcatctaggttcagcggcggtggctctgggacagatttcactctcaccatcagcagcctgcagcctgaagattttgcaagttactactgtCAACAGGCTAAGAGTTTCCCTCTTACTtttggccaggggaccaagctggagatcaa ac RVC111 ANTIBODY 201 CDRH1 aaGFS F SSY V 202 CDRH2 aa ISYDGSNK 203 CDRH3 aa ARGSGTQTPLFDY 204CDRL1 aa QSI T SW 205 CDRL2 aa D D S 206 CDRL2 long aa LIYD D S TLE 207CDRL3 aa QQY E SYSGT 208 CDRH1 nuc ggattctccttcagtagctatgtt 209CDRH2 nuc atatcatatgatggaagtaataaa 210 CDRH3 nucgcgagagggtccggaacccaaactcccctctttgactac 211 CDRL1 nuc cagagtattactagctgg212 CDRL2 nuc gatgactcc 213 CDRL2 long nuc ctgatctatgatgactccactttggaa214 CDRL3 nuc caacagtatgagagttattcagggacg 215 heavy chain aaQVQLVESGGGVVQPGRSLRLSCASGF S FSSY V MYWVRQA PGKGLEWVTIISYDGSNKYYADSVKGRFTISRDNSKNTLY LQMNSLRAEDTAVYYCARGSGTQTPLFDYWGQGTLVTVSS216 light chain aa DIQMTQSPSTLSASVGDRVTITCRAN QSI T SW VAWYQQMPGRAPKLLIYD D STLESGVPSRFSGSGSGTEFTLTISSLQP DDFATYYCQQY E SYSGTFGQGTKVEIK217 heavy chain nuc caggtgcaactggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcctctggattctccttcagtagctatgttatgtactgggtccgccaggctccaggcaaggggctggagtgggtgacaattatatcatatgatggaagtaataaatactacgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagctgaggacacggctgtctattactgtgcgagagggtccggaacccaaactcccctctttgactactggggccagggaaccctggtcaccgtctcctca g 218 light chain nucgacatccagatgacccagtctccttccaccctgtctgcatctgtgggagacagagtcaccatcacttgccgggccaatcagagtattactagctgggtggcctggtatcagcagatgccagggagagcccctaaactcctgatctatgatgactccactttggaaagtggggtcccatcaaggttcagcggcagtggatctgggacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaacagtatgagagttattcagggacgttcggccaagggaccaaggtggaaatcaa ac *the sequences highlightedin bold are CDR regions (nucleotide or aa) and the underlined residuesare mutated residues as compared to the ″germline″ sequence.

The invention claimed is:
 1. A method of preventing and/or treating aRABV and/or non-RABV lyssavirus infection in a subject, wherein themethod comprises administering to a subject in need thereof an antibody,or an antigen binding fragment thereof, that neutralizes a lyssavirusinfection, wherein the antibody or antigen binding fragment comprisesheavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences as set forth in SEQ ID NOs:165-169 and 171, respectively, or in SEQ ID NOs: 165-168 and 170-171,respectively.
 2. The method of claim 1, wherein the antibody or antigenbinding fragment comprises a heavy chain variable region having at least80% sequence identity to the amino acid sequence of SEQ ID NO:179 and alight chain variable region having at least 80% sequence identity to theamino acid sequence of SEQ ID NO:180, provided that the antibody orantigen binding fragment comprises heavy chain CDRH1, CDRH2, and CDRH3amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acidsequences as set forth in SEQ ID NOs: 165-169 and 171, respectively, orin SEQ ID NOs: 165-168 and 170-171, respectively.
 3. The method of claim1, wherein the antibody or antigen binding fragment comprises a heavychain variable region having at least 90% sequence identity to the aminoacid sequence of SEQ ID NO: 179 and a light chain variable region havingat least 90% sequence identity to the amino acid sequence of SEQ ID NO:180, provided that the antibody or antigen binding fragment comprisesheavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences as set forth in SEQ ID NOs:165-169 and 171, respectively, or in SEQ ID NOs: 165-168 and 170-171,respectively.
 4. The method of claim 1, wherein the antibody or antigenbinding fragment comprises a heavy chain variable region having at least95% sequence identity to the amino acid sequence of SEQ ID NO: 179 and alight chain variable region having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 180, provided that the antibody orantigen binding fragment comprises heavy chain CDRH1, CDRH2, and CDRH3amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acidsequences as set forth in SEQ ID NOs: 165-169 and 171, respectively, orin SEQ ID NOs: 165-168 and 170-171, respectively.
 5. The method of claim1, wherein the antibody or antigen binding fragment comprises a heavychain variable region having at least 99% sequence identity to the aminoacid sequence of SEQ ID NO: 179 and a light chain variable region havingat least 99% sequence identity to the amino acid sequence of SEQ ID NO:180, provided that the antibody or antigen binding fragment comprisesheavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences as set forth in SEQ ID NOs:165-169 and 171, respectively, or in SEQ ID NOs: 165-168 and 170-171,respectively.
 6. The method of claim 1, wherein the antibody or antigenbinding fragment is a monoclonal antibody or an antigen binding fragmentthereof.
 7. The method of claim 1, wherein the antibody or antigenbinding fragment neutralizes infection by RABV CVS-11 with an IC₉₀ of400 ng/ml or less.
 8. The method of claim 1, wherein the antibody orantigen binding fragment comprises a heavy chain variable region havingthe amino acid sequence of SEQ ID NO: 179 and a light chain variableregion having the amino acid sequence of SEQ ID NO:
 180. 9. The methodof claim 1, wherein the antibody or antigen binding fragment isaccording to gRVC58.
 10. The method of claim 1, wherein the antibody orantigen binding fragment is RVC58.
 11. The method of claim 1, whereinthe antibody or antigen binding fragment is a human antibody, amonoclonal antibody, a human monoclonal antibody, a purified antibody, asingle chain antibody, Fab, Fab′, F(ab′)2, Fv or scFv.
 12. The method ofclaim 1, wherein the antibody or antigen binding fragment neutralizeslyssavirus infection by (i) RABV and (ii) at least 50% of all isolatesof non-RABV lyssaviruses selected from the group consisting ofABLV/Australia/bat/9810AUS-1998/V1039-2011/ABLV, 98010/ABLV, 1301Bokeloh bat lyssavirus/BBLV, 86132SA/DUVV, DUVV/SouthAfrica/human/96132SA-1971/RS639-2012/DUVV,EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV1b/France/bat/8918-1989/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, 94112/EBLV-2, 02053/EBLV-2,8619/LBV, MOK/MOK, Shimoni bat Virus/SHIV, West Caucasian batVirus/WCBV, Australian bat lyssavirus/RV634/ABLV, Aravan Virus/ARAV,Duvenhage Virus RSA2006/DUVV, Duvenhage Virus ZIM86-RV 131/DUVV,European bat lyssavirus 1.RV20/EBLV-1, European bat lyssavirus1.RV9/EBLV-1, EBLV 1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, European bat lyssavirus2.RV1787/EBLV-2, European bat lyssavirus 2.RV628/EBLV-2, IrkutVirus/IRKV, Khujand Virus/KHUV, 8619/LBV, Lagos Bat Virus NIG56-RV1/LBV,Lagos Bat Virus SA2004/LBV, Mokola Virus NIG68.RV4/MOK, Mokola Virus98/071 RA36/MOK and Ikoma lyssavirus/IKOV with an IC50 of less than10000 ng/ml for ABLV/Australia/bat/9810AUS-1998/V1039-2011/ABLV,98010/ABLV, 1301 Bokeloh bat lyssavirus/BBLV, 86132SA/DUVV,DUVV/SouthAfrica/human/96132SA-1971/RS639-2012/DUVV,EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV1b/France/bat/8918-1989/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, 94112/EBLV-2, 02053/EBLV-2,8619/LBV, MOK/MOK tested as infectious viruses and with an IC90 of lessthan 10000 ng/ml for Shimoni bat Virus/SHIV, West Caucasian batVirus/WCBV, Australian bat lyssavirus/RV634/ABLV, Aravan Virus/ARAV,Duvenhage Virus RSA2006/DUVV, Duvenhage Virus ZIM86-RV 131/DUVV,European bat lyssavirus 1.RV20/EBLV-1, European bat lyssavirus1.RV9/EBLV-1, EBLV 1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, European bat lyssavirus2.RV1787/EBLV-2, European bat lyssavirus 2.RV628/EBLV-2, IrkutVirus/IRKV, Khujand Virus/KHUV, 8619/LBV, Lagos Bat Virus NIG56-RV1/LBV,Lagos Bat Virus SA2004/LBV, Mokola Virus NIG68.RV4/MOK, Mokola Virus98/071 RA36/MOK and Ikoma lyssavirus/IKOV tested as pseudotyped viruses.13. The method of claim 1, wherein the antibody or antigen bindingfragment neutralizes lyssavirus infection by at least 70% of non-RABVphylogroup I lyssaviruses selected from the group consisting of DUVV,EBLV-1, EBLV-2, ABLV, IRKV, KHUV, and ARAV, with an IC50 of less than10000 ng/ml.
 14. The method of claim 1, wherein the antibody or antigenbinding fragment neutralizes lyssavirus infection by at least 70% of theisolates of non-RABV phylogroup I lyssaviruses selected from the groupconsisting of ABLV/Australia/bat/9810AUS-1998/V1039-2011/ABLV,98010/ABLV, 1301 Bokeloh bat lyssavirus/BBLV, 86132SA/DUVV,DUVV/SouthAfrica/human/96132SA-1971/RS639-2012/DUVV,EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV1b/France/bat/8918-1989/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, 94112/EBLV-2, 02053/EBLV-2,Australian bat lyssavirus/RV634/ABLV, Aravan Virus/ARAV, Duvenhage VirusRSA2006/DUVV, Duvenhage Virus ZIM86-RV 131/DUVV, European bat lyssavirus1.RV20/EBLV-1, European bat lyssavirus 1.RV9/EBLV-1, EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, European bat lyssavirus2.RV1787/EBLV-2 and European bat lyssavirus 2.RV628/EBLV-2, IrkutVirus/IRKV, Khujand Virus/KHUV, with an IC50 of less than 10000 ng/mlfor ABLV/Australia/bat/9810AUS-1998/V1039-2011/ABLV, 98010/ABLV, 1301Bokeloh bat lyssavirus/BBLV, 86132SA/DUVV,DUVV/SouthAfrica/human/96132SA-1971/RS639-2012/DUVV,EBLV1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV1b/France/bat/8918-1989/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, 94112/EBLV-2 and02053/EBLV-2, tested as infectious viruses and with an IC90 of less than10000 ng/ml for Australian bat lyssavirus/RV634/ABLV, Aravan Virus/ARAV,Duvenhage Virus RSA2006/DUVV, Duvenhage Virus ZIM86-RV 131/DUVV,European bat lyssavirus 1.RV20/EBLV-1, European bat lyssavirus1.RV9/EBLV-1, EBLV 1a/France/bat/122938-2002/V3951-2009/EBLV-1,EBLV2/UK/bat/RV1332-2002/V3951-2009/EBLV-2, European bat lyssavirus2.RV1787/EBLV-2, European bat lyssavirus 2.RV628/EBLV-2, IrkutVirus/IRKV and Khujand Virus/KHUV tested as pseudotyped viruses.
 15. Themethod of claim 1, wherein the antibody or antigen binding fragmentneutralizes infection by EBLV-1.
 16. The method of claim 1, wherein themethod comprises post-exposure prophylaxis.
 17. The method of 1, whereinthe subject is vaccinated against RABV and/or non-RABV lyssavirusinfection.
 18. The method of claim 1, comprising administering theantibody or antigen binding fragment, up to seven days, or up to fivedays, after infection of the subject.
 19. The method of claim 1, whereinthe subject has received or is receiving: (i) a vaccine; (ii) anantiviral; (iii) interferon-alpha; (iv) ketamine; or (v) any combinationof (i)-(v).
 20. The method of claim 1, comprising administering theantibody or antigen binding fragment in a standard PEP scheme, incombination with a vaccine, or in the first treatment of the standardPEP scheme only.
 21. The method of claim 1, comprising administering theantibody or antigen binding fragment to the subject at a dose of 0.005to 100 mg/kg, at a dose of 0.0075 to 50 mg/kg, at a dose of 0.01 to 10mg/kg, at a dose of 0.01 to 1 mg/kg, or at a dose of 0.01 to 0.1 mg/kg.22. The method of claim 1, comprising administering the antibody orantigen binding fragment to the subject from 1 to 6 days, or from 2 to 5days, after infection.
 23. The method of claim 1, comprisingadministering the antibody or antigen binding fragment to the subjectwithout (i) concomitant and/or (ii) subsequent administration of avaccine.
 24. The method of claim 1, comprising administering theantibody or antigen binding fragment to the subject at a dose of 0.01 to100 mg/kg, at a dose of 0.1 to 75 mg/kg, at a dose of 1 to 60 mg/kg, orat a dose of 10 to 50 mg/kg.
 25. The method of claim 1, wherein theantibody or antigen binding fragment is a first antibody or antigenbinding fragment and the method further comprises administering to thesubject a second antibody, or an antigen binding fragment thereof, thatneutralizes a lyssavirus infection, wherein the second antibody orantigen binding fragment comprises heavy chain CDRH1, CDRH2, and CDRH3amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 amino acidsequences as set forth in SEQ ID NOs: 93-95, 96, 97, and 99,respectively, or in SEQ ID NOs: 93-95, 96, 98, and 99, respectively. 26.The method of claim 25, wherein the second antibody or antigen bindingfragment comprises a heavy chain variable region having at least 80%sequence identity to the amino acid sequence of SEQ ID NO:107 and alight chain variable region having at least 80% sequence identity to theamino acid sequence of SEQ ID NO: 108, provided that the second antibodyor antigen binding fragment comprises heavy chain CDRH1, CDRH2, andCDRH3 amino acid sequences and light chain CDRL1, CDRL2, and CDRL3 aminoacid sequences as set forth in SEQ ID NOs: SEQ ID NOs: 93-95, 96, 97,and 99, respectively, or in SEQ ID NOs: 93-95, 96, 98, and 99,respectively.
 27. The method of claim 25, wherein the second antibody orantigen binding fragment comprises a heavy chain variable region havingat least 90% sequence identity to the amino acid sequence of SEQ IDNO:107 and a light chain variable region having at least 90% sequenceidentity to the amino acid sequence of SEQ ID NO: 108, provided that thesecond antibody or antigen binding fragment comprises heavy chain CDRH1,CDRH2, and CDRH3 amino acid sequences and light chain CDRL1, CDRL2, andCDRL3 amino acid sequences as set forth in SEQ ID NOs: SEQ ID NOs:93-95, 96, 97, and 99, respectively, or in SEQ ID NOs: 93-95, 96, 98,and 99, respectively.
 28. The method of claim 25, wherein the secondantibody or antigen binding fragment comprises a heavy chain variableregion having at least 95% sequence identity to the amino acid sequenceof SEQ ID NO:107 and a light chain variable region having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 108, providedthat the second antibody or antigen binding fragment comprises heavychain CDRH1, CDRH2, and CDRH3 amino acid sequences and light chainCDRL1, CDRL2, and CDRL3 amino acid sequences as set forth in SEQ ID NOs:SEQ ID NOs: 93-95, 96, 97, and 99, respectively, or in SEQ ID NOs:93-95, 96, 98, and 99, respectively.
 29. The method of claim 25, whereinthe second antibody or antigen binding fragment comprises a heavy chainvariable region having at least 97% sequence identity to the amino acidsequence of SEQ ID NO:107 and a light chain variable region having atleast 97% sequence identity to the amino acid sequence of SEQ ID NO:108, provided that the second antibody or antigen binding fragmentcomprises heavy chain CDRH1, CDRH2, and CDRH3 amino acid sequences andlight chain CDRL1, CDRL2, and CDRL3 amino acid sequences as set forth inSEQ ID NOs: SEQ ID NOs: 93-95, 96, 97, and 99, respectively, or in SEQID NOs: 93-95, 96, 98, and 99, respectively.
 30. The method of claim 25,wherein the second antibody or antigen binding fragment comprises aheavy chain variable region having at least 99% sequence identity to theamino acid sequence of SEQ ID NO:107 and a light chain variable regionhaving at least 99% sequence identity to the amino acid sequence of SEQID NO: 108, provided that the second antibody or antigen bindingfragment comprises heavy chain CDRH1, CDRH2, and CDRH3 amino acidsequences and light chain CDRL1, CDRL2, and CDRL3 amino acid sequencesas set forth in SEQ ID NOs: SEQ ID NOs: 93-95, 96, 97, and 99,respectively, or in SEQ ID NOs: 93-95, 96, 98, and 99, respectively. 31.The method of claim 25, wherein the second antibody or antigen bindingfragment comprises a heavy chain variable region having the amino acidsequence of SEQ ID NO:107 and a light chain variable region having theamino acid sequence of SEQ ID NO:
 108. 32. The method of claim 25,wherein the second antibody or antigen binding fragment is RVC20. 33.The method of claim 25, wherein the first antibody or antigen bindingfragment and the second antibody or antigen binding fragment areadministered simultaneously or consecutively.
 34. The method of claim25, wherein the first antibody or antigen binding fragment and thesecond antibody or antigen binding fragment are comprised in a samepharmaceutical composition.